Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility

Abstract

The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosa flgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility. Fig. 1. Growth phase-dependent transcription and translation of flgZ.A. FlgZ protein levels of P. aeruginosa PAO1 grown in exponential and stationary growth phase (adjusted to the same optical density) were determined by Western blot analysis using an anti-FlgZ antibody. B. Relative fold change of flgM, flgN and flgZ gene expression in stationary growth phase compared to exponential growth phase. Data were extracted from a previous publication (Dötsch et al., 2012). RNA sequencing was performed with cultures grown to OD 1 (exponential) and for 12 h (stationary). Both were sequenced in duplicates. The obtained sequences were mapped to the PA14 genome. Absolute read counts were normalized to yield nRPK (normalized reads per kilobase of gene sequence) values. C. A set of six primers was used in different combinations to PCR-amplify intragenic (1, 2, 3) and intergenic (4, 5) regions of the flgMNZ genes as indicated. D. Reverse Transcriptase PCR: cDNA was generated from RNA isolated from exponential (left) and from early stationary (right) growth phase. Lane 1-3 correspond to the intragenic PCR products of flgM (1), flgN (2), flgZ (3), lane 4-5 to the intergenic flgMN (4) and flgNZ (5) PCR products. As negative control (NC) total RNA was not subjected to reverse transcription.

Taming the flagellar motor of pseudomonads with a nucleotide messenger

“…In the case of temporal sequestration, a subset of the DGCs/PDEs are expressed or activated in a defined temporal window; whereas in the case of spatial compartmentation, the DGCs/PDEs and the cognate c-di-GMP binding receptors are required to be located in close proximity. The observed interaction or co-localization between FlgZ, MapZ and FimW and some of their signalling partners at the flagellated pole provides some circumstantial support for compartmentalized signalling (Basu Roy and Sauer, 2014;Baker et al, 2016;Xu et al, 2016b;Bense et al, 2019;Laventie et al, 2019). The receptor-dependent functional response may also be achieved without compartmentalizing the signalling proteins.…”

“…The Pseudomonas protein FlgZ, which exhibits weak homology with the YcgR proteins of E. coli and S. Typhimurium, is encoded by a gene located immediately downstream of the flagellar genes flgM and flgN in the genomes of all known Pseudomonas species (Martínez-Graneroet al, 2014). In P. aeruginosa, the FlgZ protein is sequestered to the flagellated pole through direct interaction with the stator protein MotC at elevated c-di-GMP concentrations (Martínez-Graneroet al, 2014;Baker et al, 2016;Bense et al, 2019). The formation of the FlgZ/c-di-GMP/MotC complex results in the disengagement of the MotC/D stator units from the motor (Fig.…”

c-di-GMP-related phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein

“…FlgZ is a c-di-GMP binding effector that controls P. aeruginosa motility via its interaction with the MotCD stator. Bense et al described that FimV is involved in increasing the polar localized c-di-GMP bound FlgZ 45 , however the mechanism is still unknown and a putative contribution of DgcP is yet to be assessed.…”

A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

“…HubP/FimV orthologs occur in a number of different bacterial species (e.g., P. aeruginosa, Vibrio and Shewanella). In these species these proteins coordinate a range of different cellular processes involving, but not restricted to, flagellation, swimming motility, and chemotaxis (16)(17)(18)(19)(20)(21)(22)(23), type IV pili assembly and activity (24)(25)(26)(27)(28)(29), and recruitment of the origin of replication to the cell pole (15,16). The polar localization of PdeB solely depends on its GGDEF (DGC) domain, which directly interacts with the C-terminal FimV domain of HubP (15).…”