The GGDEF Domain of the Phosphodiesterase PdeB in Shewanella putrefaciens Mediates Recruitment by the Polar Landmark Protein HubP

Abstract

Bacteria commonly exhibit a high degree of cellular organization and polarity which affect many vital processes such as replication, cell division, and motility. In Shewanella and other bacteria, HubP is a polar marker protein which is involved in proper chromosome segregation, placement of the chemotaxis system, and various aspects of pilus- and flagellum-mediated motility. Here, we show that HubP also recruits a transmembrane multidomain protein, PdeB, to the flagellated cell pole. PdeB is an active phosphodiesterase and degrades the second messenger c-di-GMP. In Shewanella putrefaciens, PdeB affects both the polar and the lateral flagellar systems at the level of function and/or transcription in response to environmental medium conditions. Mutant analysis on fluorescently labeled PdeB indicated that a diguanylate cyclase (GGDEF) domain in PdeB is strictly required for HubP-dependent localization. Bacterial two-hybrid and in vitro interaction studies on purified proteins strongly indicate that this GGDEF domain of PdeB directly interacts with the C-terminal FimV domain of HubP. Polar localization of PdeB occurs late during the cell cycle after cell division and separation and is not dependent on medium conditions. In vitro activity measurements did not reveal a difference in PdeB phosphodiesterase activities in the presence or absence of the HubP FimV domain. We hypothesize that recruitment of PdeB to the flagellated pole by HubP may create an asymmetry of c-di-GMP levels between mother and daughter cells and may assist in organization of c-di-GMP-dependent regulation within the cell. IMPORTANCE c-di-GMP-dependent signaling affects a range of processes in many bacterial species. Most bacteria harbor a plethora of proteins with domains which are potentially involved in synthesis and breakdown of c-di-GMP. A potential mechanism to elicit an appropriate c-di-GMP-dependent response is to organize the corresponding proteins in a spatiotemporal fashion. Here, we show that a major contributor to c-di-GMP levels and flagellum-mediated swimming in Shewanella, PdeB, is recruited to the flagellated cell pole by the polar marker protein HubP. Polar recruitment involves a direct interaction between HubP and a GGDEF domain in PdeB, demonstrating a novel mechanism of polar targeting by the widely conserved HubP/FimV polar marker.

The bacterial leader peptide peTrpL has a conserved function in antibiotic-dependent posttranscriptional regulation of ribosomal genes

“…Detection of FLAG-tagged proteins transferred onto a PVDF membrane was performed with monoclonal anti-FLAG M2-peroxidase (HRP) antibodies (Sigma-Aldrich) and Lumi-Light Western Blotting Substrate Kit (Roche). FLAG-tagged FLAG-FimV Protein (Rossmann et al, 2019) was used as a positive control.…”

Chromosome Segregation Proteins as Coordinators of Cell Cycle in Response to Environmental Conditions

“…Interestingly, in Shewanella oneidensis HubP homolog was also shown to be involved in chromosome segregation. Moreover, the identified in these bacteria interaction between HubP and PdeB, phosphodiesterase that controls c-di-GMP level in the cell, may indicate the potential link between cyclic nucleotide signaling and chromosome segregation (Rossmann et al, 2019). An interesting example of a bacterium in which the oriC is not localized at the poles but rather exhibits subpolar localization is M. xanthus.…”

New Twists and Turns in Bacterial Locomotion and Signal Transduction

“…There are over 50 potential c-di-GMP synthesis/degradation proteins in S. putrefaciens. Anna Pecina (Kai Thormann lab, Justus Liebig University Giessen) presented findings on the function of the PdeB protein, a dual domain DGC-PDE with two transmembrane segments and a periplasmic domain, which exerts a dominant PDE activity (77). This protein is localized to the flagellar pole by the polar landmark protein HubP, but, surprisingly, its dominant regulatory effect is on the lateral flagella through two likely discrete mechanisms: at the transcriptional level, presumptively mediated through the c-di-GMP-responsive transcription factor FlrA2, and at the posttranscriptional level mediated through a FlgZ-like flagellar brake, with a PilZ domain that binds c-di-GMP and specifically slows down flagellar rotation of the lateral but not the polar system.…”