| Myxococcus xanthus is a gram-negative δ-proteobacterium that utilizes the gliding motility move on the solid surfaces. The Type IV pilus-mediated motility in M. xanthus is known as social (S) motility (1). This is distinct from the adventurous (A) gliding motility of M. xanthus which is powered by an independent and different motility machinery. T4P in rod-shaped bacteria such as M. xanthus are mostly localized to one of the two cell poles. Their retraction pulls a cell forward in M. xanthus S motility and in the twitching motility of the y-proteobacterium Pseudomonas aeruginosa, the β-proteobacteria Neisseria meningitidis and Neisseria gonorrhoeae among other bacterial species (2,3). It is noteworthy that the T4P is the strongest among known biological motors as a single T4P can produce a stall force well over100pN when it retracts. M. xanthus S motility additionally requires extracellular polysaccharides (EPS) to function because M. xanthus EPS-mutants are defective in S motility. The current model postulates that a T4P is triggered to retract at its cell proximal end when its distal end binds to EPS that are either associated with the M. xanthus cell surface or deposited on the gliding substratum. About a dozen pil genes are required for T4P to function as a motor, for example, there have been various reports proposing a T4P IM complex consisting of PilM, PilN, PilO and PilP in P. aeruginosa and Neisseria. How T4P proteins form a multicomponent machine for its motor function remains an active area of scientific inquiry. M. xanthus provides a good model for T4P investigations because its social (S) gliding motility is powered by T4P. Besides its motor function in S motility, T4P had been shown to regulate EPS production in M. xanthus. In this study, the interactions among M. xanthus T4P proteins were investigated using genetics, yeast two/three-hybrid and bacteria two hybrid methods. More recently, a suppressor mutation in pilB was discovered that was capable of restoring EPS production to a pilA deletion mutant. An alternative genetic approach was explored to examine if T4P proteins form an integrated structure in M. xanthus. To examine if pilB suppressor could suppress the EPS-phenotype resulting from other T4P-mutations, the pilB+allele was replaced by pilB*(pilB site mutant) in the deletion mutants, respectively. Our genetic analysis suggests that there is an integrated T4P structure that crosses the inner membrane (IM), periplasm and the outer membrane (OM). The analysis of genetic suppression here suggested an integrated T4P structure consisting of PilB, PilC, PilM, PilN, PilO, PilP and PilQ in vivo. This integrated structure would include PilQ in the OM, the TM proteins PilN, PilO and PilC as well as the lipoprotein PilP on the IM. The cytoplasmic proteins PilM and PilB may associate with this structure dynamically as indicated by the genetic suppression patterns by pilB*. Further investigation indicated that the interactions among these proteins in M. xanthus apparently occur in the absence of the pilus filament as pilB*suppresses pilA deletion mutant but none of the other pil deletions. A systematic Y2H survey provided evidence for direct interactions among IM and OM proteins exposed to the periplasm. The above model enviasions an integrated T4P structure with extensive protein-protein interactions that were investigated more directly using Y2H system. We not only examined potential inner membrane complex but also examined all the key T4P protein interaction by Y2H. A protein or its domain is fused to the C-termini of both the GAL4activation domain (GAD) and DNA binding domain (GBD) in pGAD and pGBD vectors, respectively. The Y2H mating protocol (See Materials and Methods) was utilized here since it can be used to examine interactions among large numbers of proteins and their domains. A systematic Y2H analysis detected the following pairwise interactions among M. xanthus Pil proteins:1) PilB-PilB. It also found that PilBN interacted with PilBC. Further one mutant in pilB walker A and another mutant in pilB walker B also interacted with itself and truncated sequence of mutants interacted with the C terminal of PilB. The strengths of two truncated sequence of mutant interactions are lower than wild type.2) PilC-PilC. It is worthy to know that the interaction of PilC likely requires both N-termini and C-termini in the same plasmids at same time. The reason is that the simple PilCN doesn’t interact with PilCC, the plasmid contains N-termini and C-termini of PilC by a linker interact with itself as well as the fusion protein of C-termini and C-termini of PilC linked by a linker. These results indicated that PilC needs N-termini and C-termini exist in the same protein, C-termini of PilC likely form a dimer or both N-termini and C-termini of PilC form a dimer.3) PilQ-PilQ, PilP-PilQ, PilP-PilOå'ŒPilO-PilN and interactions between truncated proteins. Since PilP is an IM lipoprotein and PilN as well as PilO are integral IM proteins, these interactions allow the OM protein PilQ to communicate with the IM T4P proteins. PilM, while cytoplasmic, is likely anchored to the IM by binding to the cytoplasmic tail of PilN. Even though it didn’t find in M. xanthus, we still believe this interaction exist, because PilM, while cytoplasmic, is likely anchored to the IM by binding to the cytoplasmic tail of PilN in P. aeruginosa and N. meningitides. Using the same Y2H system, we extended the above interactions to T4P proteins from the non-proteobacterium T. thermophilus. Similar interactions among the T4P proteins of the non-proteobacterium Thermus thermophiles were fund by Y2H. It is especially noteworthy that TtPilW shows interactions with the same T4P proteins as PilP even though they share no detectable structural similarity in silico. Interestingly, the more detailed interactions of TtPilW and TtPilQ resembles those of GspC and GspD in T2SS instead of M. xanthus PilP and PilQ. That is, both PilW and GspC interact with the NO subdomains of their partner secretin. Similar observations were made recently between PilP and PilQ in P. aeruginosa and N. meningitides. The results indicated similar protein-protein interactions in the T4P system of this non-proteobacterium despite significant sequence divergence between T4P proteins in T. thermophilus and M. xanthus. The observations here support the model of an integrated T4P structure in the absence of a pilus in diverse bacterial species.Myxobacteria exhibit complex social traits during which large populations of cells coordinate their behaviors. A hallmark myxobacterial behavior is their ability to organize the movements of thousands of cells to build a fruiting body in which many of the cells differentiate into environmentally resistant spores. Myxobacteria are also noted for other multicellular behaviors such as swarming, rippling, elasticotaxis and predation (4). M. xanthus cells do not swim; instead these terrestrial microbes attach to surfaces and move by gliding. When these cells are grown in a static cell culture they form a biofilm on a plastic surface. Upon replacement of nutrient-rich media with starvation buffer, the cells aggregate and then erect a mound that develops into a fruiting body. From these micrographs it is apparent that cell behaviors are synchronized and coordinated to allow successful completion of their predetermined developmental program. Myxococcus fulvus HW-1is a typical salt-tolerant marine strain, which was isolated from a coastal sample in the19th century. It showed diversity comparing with classic soil bacteria. When cultured in a low concentration of saltwater, the strain exhibits a culture phenotype similar to soil myxobacterial strains, such as developing fruiting bodies and myxospores in response to starvation on solid surfaces. However, when the saltwater concentration is increased, the strain changes its living pattern by forming myxospores directly from vegetative cells (5). These characteristics make HW-1an ideal model for studying the adaptation mechanisms of salt-tolerant myxobacteria to marine environments. The part of similarity between M. xanthus and HW-1is a good way to understand the life style of HW-1under lower salt condition. The unqiue characteristic of HW-1make it as a identical model to study the relationship between behavioral shifts and the adaption to oceanic conditions. The HW-1strain was randomly mutagenized using transposon insertion, producing a dispersed-growing mutant, designated YLH0402. The mutant did not develop fruiting bodies and myxospores, was deficient in S-motility, produced less extracellular matrix and was less salt tolerant. The YLH0402strain was determined to be mutated by a single insertion in a gene of predicted Na+/H+antiporter, which has high similarity of homology genes in M. xanthus. Total seven genes, the homology genes and closed downstream and upstream genes, are co-transcription, this results indicated seven genes likely coordinated to function properly. The insertion mutant in the homolog genes that coded predicted Na+/H+antiporter protein resulted the decrease of sporulation and irregulation of fruiting body. But each single genes of this seven genes cluster didn’t show significant decrease in sporulation, the whole cluster knock-out mutant showed dramatically decrease in sporulation and normal amount of spores, this indicated that this cluster is play an important roles in the formation of spores. The results here are indicated that this cluster is an important part in maintaining the ability of response to salt and the multiple fruiting body structure. In other words, this cluster likely showed its impact in the shifts mechanisms in response the environments changed, such as the shifts between freshwater and seawater. In the base of the expressed changed outer membrane genes from the microarray experiments that detected genes transcription changed underwent the seawater replaced freshwater. Those distinct expression genes have highly homology with the corresponding genes in M. xanthus. We constructed the knock-out and insertion mutants using M. xanthus homology genes due to the immature mechanism of HW-1transformation system. These mutants we created showed important role in social behavioral and salt tolerant. In the summary, microarray and random insertion mutant results indicated that in M. fuluvus HW-1, it is likely a stress response of genome expression. That is, there are likely minus divergence between M. fuluvus HW-1and M. xanthus genomes. The difference of regulation and response model result in the unqiue gene expression approach is the key that gene function of M. fuluvus HW-1is more sensitive than genes of M. xanthus, such as the different role of predicted Na+/H+antiporter cluster between M. fuluvus HW-1and M. xanthus. |
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