Datensatz

Zusammenfassung

Myxococcus xanthus is a model organism for studying social behaviors and cell differentiation in bacteria. Upon nutrient depletion, M. xanthus cells initiate a developmental program that culminates in formation of spore-filled fruiting bodies and peripheral rods outside of fruiting bodies. Completion of this developmental program depends on an increased level of c-di-GMP. c-di-GMP is a ubiquitous signaling molecule that regulates diverse processes in bacteria including biofilm formation, synthesis of exopolysaccharides and adhesins, motility, cell cycle progression and development. DmxB is a diguanylate cyclase (DGC) that is essential for development and is responsible for the increase in c-di-GMP accumulation during development. PmxA is an active phosphodiesterase (PDE) of the HD-GYP domain type and is also important for fruiting body formation and sporulation. Here, we show that the developmental defects in a ∆pmxA mutant are restored by complementation with wild-type (WT) PmxA but not by an enzymatically inactive PmxA variant. Lack of PmxA did not measurably alter the global cellular pool of c-di-GMP and c-di-GMP accumulated at similar levels in aggregating cells and peripheral rods of both WT and ∆pmxA strains suggesting that PmxA may contribute to a local, intracellular pool of c-di-GMP. PmxA was neither important for exopolysaccharide accumulation nor for type IV pili formation in developing cells, two processes known to be regulated by c-di-GMP in M. xanthus during growth. ∆pmxA mutant cells showed hyper-agglutination and hyper-adhesion. Global transcriptomic analysis of developing WT and ∆pmxA cells revealed that 189 genes were differentially expressed in the ∆pmxA mutant and with 69 and 120 expressed at lower or higher levels, respectively in the mutant. Interestingly, among 69 genes expressed at a reduced level, nine encode Ser/Thr protein kinases suggesting a link between c-di-GMP regulation and signaling by such kinases during development. To understand regulation by c-di-GMP during development, we analyzed the expression of genes encoding proteins likely involved in c-di-GMP metabolism and regulation. Global transcriptomic analysis demonstrated that nine out of 66 such genes were highly induced during development. Using published ChIP-Seq data, we found that 18 genes (including dmxB and pmxA) were candidates for being regulated directly by the CRP-like transcriptional factor MrpC, which is a master regulator of development. We demonstrate that MrpC directly binds to the pmxA and dmxB promoters and regulates their activity positively and negatively, respectively. To further our understanding of how effects of changing levels of c-di-GMP are implemented in M. xanthus, we systematically generated in-frame deletion mutations in all genes encoding PilZ domain proteins and analyzed their role in motility and development. We identified two PilZ-domain proteins (MXAN_1087 and MXAN_2604) that regulate the reversal frequency in T4P-dependent and gliding motility.