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

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 Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

Corrigendum: The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

[This corrects the article DOI: 10.3389/fmicb.2021.668892.].

Isolation and Characterization of Shewanella Phage Thanatos Infecting and Lysing Shewanella oneidensis and Promoting Nascent Biofilm Formation.

Species of the genus Shewanella are widespread in nature in various habitats, however, little is known about phages affecting Shewanella sp. Here, we report the isolation of phages from diverse freshwater environments that infect and lyse strains of Shewanella oneidensis and other Shewanella sp. Sequence analysis and microscopic imaging strongly indicate that these phages form a so far unclassified genus, now named Shewanella phage Thanatos, which can be positioned within the subfamily of Tevenvirinae (Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Caudovirales; Myoviridae; Tevenvirinae). We characterized one member of this group in more detail using S. oneidensis MR-1 as a host. Shewanella phage Thanatos-1 possesses a prolate icosahedral capsule of about 110 nm in height and 70 nm in width and a tail of about 95 nm in length. The dsDNA genome exhibits a GC content of about 34.5%, has a size of 160.6 kbp and encodes about 206 proteins (92 with an annotated putative function) and two tRNAs. Out of those 206, MS analyses identified about 155 phage proteins in PEG-precipitated samples of infected cells. Phage attachment likely requires the outer lipopolysaccharide of S. oneidensis, narrowing the phage's host range. Under the applied conditions, about 20 novel phage particles per cell were produced after a latent period of approximately 40 min, which are stable at a pH range from 4 to 12 and resist temperatures up to 55°C for at least 24 h. Addition of Thanatos to S. oneidensis results in partial dissolution of established biofilms, however, early exposure of planktonic cells to Thanatos significantly enhances biofilm formation. Taken together, we identified a novel genus of Myophages affecting S. oneidensis communities in different ways.