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Abstract

In response to nutrient starvation, the soil bacterium Myxococcus xanthus initiates a complex developmental program resulting in the formation of fruiting bodies inside which the rod-shaped motile cells differentiate into environmentally resistant, spherical and diploid myxospores. Some cells, referred to as peripheral rods, remain as rod-shaped haploid cells outside of fruiting bodies. Replication is essential for fruiting body formation and sporulation. During vegetative conditions, cells grow asynchronously with respect to the cell cycle. The cells contain one to two chromosomes and initiation of replication proceeds immediately after cell division. Here we investigated the mechanism underlying the formation of diploid spores. We confirmed the diploid chromosome content of mature spores by single cell and cell population analysis. Our results indicated that peripheral rods are cells in various stages of the cell cycle that are able to replicate. To address regulation of cell division, we focused on the key cell division protein FtsZ and its regulators PomX, PomY and PomZ. Experiments in which future myxospores and future peripheral rods were separated demonstrated that the protein levels of these four proteins decreased during development specifically in cells dedicated to become spores. In line with this, we showed that the presence of each of these proteins is not essential for fruiting body formation and sporulation. Transcriptional analysis revealed a decrease in transcript numbers of ftsZ, pomX, pomY and pomZ upon starvation. Moreover we observed that FtsZ is constitutively degraded during vegetative growth as well as during development and has the same half-life under these two conditions. We conclude that FtsZ proteolysis outperforms FtsZ synthesis during development resulting in elimination of FtsZ specifically in future spores. To address whether the decrease in FtsZ level in future spores causes the inhibition of cell division in these cells, we expressed ftsZ constitutively in developing cells. Remarkably, many of the spores formed by these cells contained one chromosome. Thus, the elimination of FtsZ in cells destined to become spores inhibits cell division and guarantees the formation of diploid myxospores. Most likely the ATP-dependent protease LonD is indirectly involved in FtsZ turnover but is not responsible for the transcriptional downregulation upon starvation. Interestingly, sporulation did not depend on chromosome content however, chromosome content affects spore morphology. Cells which enter the developmental program with a chromosome content higher than WT cells form spores that are increased in average size and chromosome content. Although the decrease in FtsZ protein levels and ftsZ transcript numbers is specific to starvation, it seems to be independent of RelA and the stringent response. Additionally, regulation of FtsZ levels is independent of the global pool of the second messenger cyclic di-GMP which was shown to be an essential regulator of multicellular development in M. xanthus.