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After fixation, cells were centrifuged at 5,200 and washed once with 600 L PBS

After fixation, cells were centrifuged at 5,200 and washed once with 600 L PBS. the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle development and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator BAY 293 cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory BAY 293 histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulate to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle. In multicellular organisms, cellular differentiation is required for the formation of complex tissues and organs (1). In unicellular organisms, the ability to coordinate and control specialized cell morphologies and functions is critical for niche survival in diverse environments (2). exhibits a dimorphic life cycle where asymmetric division results in the production of a nonreproductive, motile swarmer cell and a reproductive, nonmotile stalked cell. In addition to their distinct reproductive states, each of these cell types possesses different polar structures. The swarmer cell is equipped with a single flagellum and multiple type IVc tight adherence (tad) pili at the same pole that are lost upon cellular differentiation into the stalked cell. Tad pili are subsequently replaced Mouse monoclonal to CD59(PE) with a holdfast adhesin that mediates irreversible surface attachment and a thin cell-envelope extension called the stalk (3, 4). Distinguishing characteristics between swarmer and stalked cells are partly due to the action of the grasp response regulator CtrA (4). In swarmer cells, CtrA is usually phosphorylated and binds strongly to chromosomal sites near the origin of replication, preventing the initiation of DNA replication and thus locking cells in a nonreproductive, arrested G1 phase. During differentiation from swarmer to stalked cell, CtrA is usually dephosphorylated and proteolytically cleaved to allow for entry into S phase and subsequent chromosome replication (4). Regulatory control over differentiation is usually mediated by oscillating levels of cyclic diguanylate monophosphate (c-di-GMP), a ubiquitous secondary messenger molecule that coordinates bacterial behavior in diverse species (5). Newborn swarmer cells have low concentrations of c-di-GMP that slowly increase as they age. Between 20 and 40 min postdivision, a maximal level of c-di-GMP is usually observed, coinciding with holdfast synthesis and the transition from the motile to the sessile state. At the same time, a high level of c-di-GMP stimulates the dephosphorylation and deactivation of CtrA, allowing for chromosome replication as the swarmer cell differentiates (4). C-di-GMP levels are controlled by the activity of the two histidine kinases PleC and DivJ, which localize at the swarmer and stalked pole of predivisional cells, respectively, and which dictate the distinct fates of the two progeny cells (6). Delocalization of PleC and localization of DivJ at the incipient stalked pole during cell differentiation mediate the activation of the diguanylate cyclase PleD by phosphorylation, resulting in an increase in c-di-GMP. Although the signal transduction network governing the transition from swarmer to stalked cell has been well-described, whether surface attachment impacts this process is not known. Here, we demonstrate that inhibition of dynamic pilus activity stimulates c-di-GMP to initiate stalked cell development. We show that physical obstruction of pilus retraction and surface contact stimulates the initiation BAY 293 of DNA replication. We show that a mutation in the outer-membrane pilus secretin CpaC that partially disrupts pilus retraction stimulates holdfast synthesis and initiation of DNA replication in a PilA-dependent fashion. Finally, we show that physical obstruction of pilus retraction directly stimulates c-di-GMP synthesis by accelerating the delocalization of PleC and localization of DivJ at the incipient stalked pole, key actions in the activation of PleD and the production of c-di-GMP. Thus, by stimulating the synthesis of the holdfast (7) and cell differentiation, surface contact ensures that the permanently attached cell BAY 293 enters the stalked phase, which is BAY 293 best adapted for nutrient uptake on a surface (8). Results Obstruction of Pilus Retraction Stimulates DNA Replication Initiation. Whether mechanical inputs can stimulate cell differentiation is usually unknown. Previous work has exhibited that swarmer cells produce holdfast in response to surface contact impartial of cell age (7, 9, 10), and recent findings suggest that this surface-stimulated holdfast synthesis.