Dokument

Summary:
Myxococcus xanthus is a representative of Gram-negative bacteria that are able to form quiescent, environmentally-resistant spores in response to changes in environmental conditions, such as nutrient depletion. M. xanthus spores are formed inside fruiting bodies as final stage of an elaborate developmental program. In contrast to well studied Gram-positive spore formers, where sporulation is linked to cell division, M. xanthus spores are formed by rounding up of an entire rod-shaped cell. Studies on the core sporulation process in M. xanthus are impeded by the complexity of the starvation induced developmental program, subpopulations within a developing colony, the low proportion of cells that convert into spores, and their high mechanical resistance. We took advantage of the glycerol induced spore formation process and performed micro array analysis. This study revealed that 1,596 genes are significantly at least two-fold up- or down-regulated within four hours after addition of the inducer. Most of the genes that previously have been identified to play a role during starvation induced sporulation were found to be up-regulated indicating that the glycerol induced sporulation is an appropriate model to study the core sporulation process in M. xanthus. The array data analysis led to identification of a novel and highly up-regulated genomic locus termed nfs (necessary for sporulation). The nfs locus encodes for eight proteins that show no homology to characterized proteins. Bioinformatics, mutational and immunoblot analysis suggest that the Nfs-proteins localize to the cell envelope and form a complex. In-frame deletion of the nfs-genes led to a severe defect both in glycerol and starvation induced sporulation, whereas aggregation was not affected. In response to glycerol induction, the ∆nfs mutant displayed transiently aberrant cell morphology. Transcription from the nfs-promoter was detectable 30 minutes after induction with glycerol. Translational fusion of the putative promoter region to mcherry as reporter suggests that the nfs-genes only accumulate in spores. Analysis of nfs-expression in developmental mutant backgrounds suggests that nfs-expression is dependent on C-signaling and controlled by FruA. Based on this observation it is hypothesized that the Nfs proteins are specifically expressed during spore formation, that they form a cell envelope-associated complex and that they play a crucial role in generating viable spores. In addition, the role of the filament-forming cytoskeletal protein MreB was analyzed in respect to spore formation using genetic and biochemical approaches. MreB plays a crucial role in maintaining a rod-like cell shape in most known rod-shaped bacteria and has been shown to be a key-organizer of cell wall synthesis. The results suggest that the protein stays present during glycerol induced spore formation but becomes degraded during starvation induced spore formation suggesting two alternative ways of sphere formation in M. xanthus. Assays of spore germination with the MreB perturbing compound A22 revealed that MreB polymerization is an important precondition for germination of spherical spores, i.e. when cells re-establish rod-like cell morphology. The results of this work could provide a set of tools that can be used to reveal fundamental mechanisms of cell shape determination and coordinated cell shape conversion during spore formation as well as peptidoglycan synthesis with respect to cell morphogenic events in Gram-negative bacteria.

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