PflI, a Protein Involved in Flagellar Positioning in Caulobacter crescentus

Abstract

The bacterial flagellum is important for motility and adaptation to environmental niches. The sequence of events required for the synthesis of the flagellar apparatus has been extensively studied, yet the events that dictate where the flagellum is placed at the onset of flagellar biosynthesis remain largely unknown. We addressed this question for alphaproteobacteria by using the polarly flagellated alphaproteobacterium Caulobacter crescentus as an experimental model system. To identify candidates for a role in flagellar placement, we searched all available alphaproteobacterial genomes for genes of unknown function that cluster with early flagellar genes and that are present in polarly flagellated alphaproteobacteria while being absent in alphaproteobacteria with other flagellation patterns. From this in silico screen, we identified pflI. Loss of PflI function in C. crescentus results in an abnormally high frequency of cells with a randomly placed flagellum, while other aspects of cell polarization remain normal. In a wild-type background, a fusion of green fluorescent protein (GFP) and PflI localizes to the pole where the flagellum develops. This polar localization is independent of the flagellar protein FliF, whose oligomerization into the MS ring is thought to define the site of flagellar synthesis, suggesting that PflI acts before or independently of this event. Overproduction of PflI-GFP often leads to ectopic localization at the wrong, stalked pole. This is accompanied by a high frequency of flagellum formation at this ectopic site, suggesting that the location of PflI is a sufficient marker for a site for flagellar assembly.Flagellar arrangement has long been used as a defining characteristic in bacterial taxonomy. Bacteria can have flagella emerging from a specific site, such as the cell pole, or from seemingly random positions along the cell body (as in peritrichously flagellated bacteria). Differences in flagellar distribution can be important for how various species adapt to specific environments. While the biosynthesis of the flagellum and the temporal regulation of its assembly have been extensively studied (5, 32), we know little about what controls the cellular position of flagellar assembly. The distribution of flagella in peritrichous bacteria may be random and stochastically driven, but spatial mechanisms must exist in polarly flagellated bacteria to ensure that flagellar assembly takes place at the right location.It has been shown that in several polarly flagellated species of gammaproteobacteria, the flhF gene product is necessary for restricting the flagellum to a polar position (38, 41). flhF encodes a GTP-binding protein homologous to components of the signal recognition particle (SRP) pathway; in its absence, Pseudomonas putida and Pseudomonas aeruginosa have reduced motility and randomly placed flagella on the cell surface (25,38,41). Surprisingly, flhF homologs are also found in species with multiple, randomly placed flagella, indicating that FlhF is not a defining component of polarly f...

Protein localization and dynamics within a bacterial organelle

“…3F). Remarkably, a chimera of GFP to the C terminus of the TM domain (TM-GFP) from the flagellar positioning factor PflI (27) was also observed in the stalk (Fig. 3F).…”

De- and repolarization mechanism of flagellar morphogenesis during a bacterial cell cycle

“…Indeed, flagella are mispositioned in the absence of tipN, indicating that TipN is required for the proper placement, but not the assembly, of the flagellum (Huitema et al 2006;Lam et al 2006). A flagellar misplacement defect has also been observed in mutants lacking the PflI positioning factor, a bitopic membrane protein with a prolinerich domain at the C terminus facing the cytoplasm that is localized to the future flagellated pole before the synthesis of early flagellar components (Obuchowski and Jacobs-Wagner 2008). While the spatial relationship between TipN and PflI remains unexplored, TipN recruits TipF, a polytopic membrane protein that positively regulates flagellum biogenesis (Huitema et al 2006).…”

“…PflI is required for proper positioning of the flagellum and is recruited to the future flagellum assembly site before flagellar structural proteins are expressed by an unknown mechanism (Obuchowski and Jacobs-Wagner 2008). Like TipF, PflI is still polar in the absence of the MS ring protein FliF, raising the possibility that PflI localization is dependent on TipF.…”

“…We found that TipF is a receptor for c-di-GMP that peaks at the G1 / S transition and show that TipF•c-di-GMP seeds polar flagellar assembly by recruiting the PflI placement factor (Obuchowski and Jacobs-Wagner 2008) and components of the flagellar switch into a complex with TipN, a landmark protein that is prepositioned at the newborn pole (Huitema et al 2006;Lam et al 2006). Importantly, we found that c-di-GMP-induced stabilization and polarization of TipF is amplified with the coordinated transcription of the tipF gene and thus is tuned to other c-di-GMP-dependent developmental events that occur at the G1 / S transition (Paul et al 2004(Paul et al , 2008Duerig et al 2009).…”

Getting in the Loop: Regulation of Development inCaulobacter crescentus

“…A better candidate is the protein PflI. pflI mutants have a 4-to 5-fold increase in mislocalized flagella, while other polar markers are unaffected (176). PflI localizes to the swarmer pole prior to establishment of the MS ring.…”