During the developmental process of the Gram-negative soil bacterium Myxococcus xanthus, vegetatively growing rod cells differentiate to ultimately become metabolically quiescent and environmentally resistant myxospores encased within fruiting bodies. This program, initiated by nutrient deprivation, is propagated by both cell-autonomous and cell-nonautonomous signals. Our goal was to determine whether M. xanthus, like many other developmental systems, uses cell-cycle cues to regulate and control its developmental program. To address this question, the DNA replication cycle was used as a marker to monitor progression through the cell cycle in vegetative, stationary, and developing M. xanthus populations. Using flow cytometry, quantitative fluorescence microscopy, and FISH to establish the chromosome copy number of myxospores, it was determined that vegetatively growing cells contain one to two copies of the genome, but upon entry into stationary phase, the chromosome copy number drops to a single copy. Of particular interest, fruiting body-derived myxospores contain a specific two-chromosome DNA complement with both origin and terminus regions localized to the periphery of the myxospore. We speculate that this duplication of genetic information in the myxospore would help assure viability during germination by providing a second copy of each gene. The results of this study imply that not only is DNA replication tightly regulated during the developmental process of M. xanthus, but that there are also regulatory mechanisms to ensure that all myxospores acquire two copies of the chromosome.development ͉ chromosome copy number ͉ myxospore

Previous studies have demonstrated that fruiting body-derived Myxococcus xanthus myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.Myxococcus xanthus is a social gram-negative soil bacterium that when challenged with nutrient limitation enters a complex, multicellular developmental process that culminates in the formation of metabolically quiescent myxospores encased in a macroscopic fruiting body. Development is initiated by starvation for nitrogen, carbon, or phosphate and begins with a fairly homogenous distribution of cells. As development progresses, cells begin to aggregate and form dynamic aggregation centers. Eventually, these aggregation centers stabilize and recruit additional cells to become mounds, which darken into mature dome-shaped fruiting bodies containing environmentally resistant myxospores (Fig. 1).As expected from such a highly complex process, M. xanthus cells have evolved a complex regulatory network which controls motility, behavior, and temporal gene expression. Of particular interest is the coordination of chromosome replication and the cell cycle in relation to development. We have recently reported that development in M. xanthus specifically produces myxospores, which each have two fully replicated copies of its single 9.1-Mbp chromosome (23,24). From these observations, it is apparent that the possession of two chromosomes is the favored state for chromosome copy number in the myxospore. However, is this state necessary or essential for the process of development? This question is relevant because unlike sporulation in other developmental prokaryotes, such as Bacillus subtilis, which requires a cell division event to produce a single spore (13), each M. xanthus cell has the capacity to differentiate into a myxospore without the necessity of a cell division event (4).In this study, we sought to use known chemical inhibitors of DNA replication to observe their effects on the developmental process in M. xanthus. The goal of this study was not only to determine if concurrent DNA replication is essential for progression through the developmental program, but to determine the timing of this event in relation to the M. xanthus developmental program. MATERIALS AND METHODSBacterial growth and media. M. xanthus was grown at 33°C in CTTYE liquid medium or on CTTYE containing 1.5% agar (1). Nalidixic acid, hydroxyurea, or novobiocin was added as described below. Nalidixic acid and novobiocin were used, each at a concentration of 20 g/ml (10) (and 40 g/ml for genetic selections), while ...

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