The role of Protein W in the process of Myxoccocus xanthus sporulation

The Gram negative Myxoccocus xanthus bacteria provide an ideal model system for the understanding of social behavior in prokaryotes, as these bacteria respond to stress signals by social communication, Upon starvation, M. xanthus cells aggregate to form fruiting bodies within which they differentiate into myxospores, while remaining cells disintegrate, thus serving as a nutrient source for their sporulating siblings. The sporulation mechanism of M. xanthus cells differs significantly from the mechanism revealed by Gram- positive spore-forming bacteria, such as Bacillus subtilis. In contrast to sporulation in B. subtilis where the spore develops within the mother cell that ultimately disintegrates, in M. xanthus, the whole cell undergoes compaction to ultimately form a spore. Moreover, the chromosome size of M. xanthus is ∼2.2 times larger than that of B. subtilis and myxospores contain two chromosomal copies, whereas only one chromosomal copy is regularly present in spores derived from other organisms. These observations mean that the overall amount of DNA in myxospores is ∼4.4 times larger than in other spores. This large DNA complement must be tightly packed within the spore core and effectively protected. Although the global morphological changes that occur during M. xanthus sporulation have been documented, little is known about the corresponding molecular changes that allow cells inside fruiting bodies to differentiate and evolve into stress-resistant myxospores. Even though it is likely that M. xanthus uses a large number of proteins to construct a myxospore, currently only few proteins known to play roles in the process of spore development have been identified.;Accordingly, our studies were aimed to characterize a novel protein, termed protein W (PrW), which is a major myxospore protein. Our studies indicate that PrW displays unique structural and biochemical characteristics, as well as large-scale effects on the morphology of the fruiting bodies. PrW was purified from M. xanthus rnyxospores and was found by retardation assays to bind specifically to supercoiled DNA, in the presence of doubly charged cations, such as Mg2+. These results imply that PrW may act as a DNA-binding protein in M. xanthus rnyxospores, and that this interaction depends upon DNA tertiary conformation. Furthermore, the requirement for Mg2+ ions may indicate that DNA-binding is promoted by ion bridges, as previously shown for another DNA-binding protein (Dps).;Secondary structural predictions as well as CD analysis indicated that the conformation of PrW is mainly α-helical. Structure-function evaluation and modeling using several methods revealed a unique repetitive motif and a highly ordered structure. All secondary structure evaluation algorithms employed on the PrW sequence predicted seven unusually long α-helices, connected through short random-coils segments. The predicted seven helices display very high internal homology, which originates from repetitive patterns that are found throughout the sequence. The helices appear to adopt a novel right-handed tetrad motif. We propose that interactions between the long helices produce a coiled-coil conformation that would be very tight, stable and ordered. Such an ordered structure can act as a template for DNA packaging within the myxospore. Support for a function of PrW as a template for packaging is obtained from our observations that the purified protein forms ordered lamellar structures, as well as from experiments conducted on E. coli cells over-expressing PrW, which implied a PrW-mediated DNA packaging.;PrW displays a substantial effect not only in the context of the individual myxospore through its interaction with the spore DNA, but also in the context of the whole fruiting body. While the WT strain forms dome shaped, elevated fruiting bodies, fruiting bodies derived from a PrW null mutant are flat and very elongated. In addition, the mutant fruiting bodies are less condensed in comparison with the densely-packed WT fruiting bodies.;Our observations support the notion that that the core protein PrW is involved in the packaging of the unusually large amount of DNA within the M. xanthus myxospore through either forming bridges between DNA double helices or through acting as a highly ordered template. The conspicuous effect on the global structure of M. xanthus fruiting bodies that we report might be related to PrW-induced DNA packaging.