Regulation of the Single Polar Flagellar Biogenesis

Abstract

Some bacterial species, such as the marine bacterium Vibrio alginolyticus, have a single polar flagellum that allows it to swim in liquid environments. Two regulators, FlhF and FlhG, function antagonistically to generate only one flagellum at the cell pole. FlhF, a signal recognition particle (SRP)-type guanosine triphosphate (GTP)ase, works as a positive regulator for flagellar biogenesis and determines the location of flagellar assembly at the pole, whereas FlhG, a MinD-type ATPase, works as a negative regulator that inhibits flagellar formation. FlhF intrinsically localizes at the cell pole, and guanosine triphosphate (GTP) binding to FlhF is critical for its polar localization and flagellation. FlhG also localizes at the cell pole via the polar landmark protein HubP to directly inhibit FlhF function at the cell pole, and this localization depends on ATP binding to FlhG. However, the detailed regulatory mechanisms involved, played by FlhF and FlhG as the major factors, remain largely unknown. This article reviews recent studies that highlight the post-translational regulation mechanism that allows the synthesis of only a single flagellum at the cell pole.

Keywords: ATPase; FlaK; FlhF; FlhG; GTPase; HubP; SflA; polar flagellum; protein localization.

Figure 1

The flagellum is a bacterial motility organ. (a) Schematic of the polar flagellar motor of Vibrio alginolyticus. The rotor-stator interaction that couples with sodium ion influx through the stator channel generates motor torque. OM, outer membrane; IM, inner membrane; PG, peptidoglycan layer. (b) The number and location of flagella vary among bacterial species.

Figure 2

FlhF and FlhG regulate the number of polar flagella in Vibrio alginolyticus. (a) Electron micrographs of V. alginolyticus strain VIO5 (wild-type for polar flagellation) and KK148 (flhG mutant of VIO5). (b) The mutation site of KK148 (upper panel) and flagellar organisation of FlhF and FlhG variants of Vibrio alginolyticus (lower panel). The mutation was mapped on flhG (Q109Amber), which forms an operon with flhF. FlhF and FlhG work antagonistically. Their overproduction or deletion/depletion confers opposite phenotypes in Vibrio alginolyticus. (c) Model for the regulation of polar flagella by FlhF and FlhG proposed in [34]. In this model, FlhF localizes at the cell pole and determines the location of flagellation. FlhG interacts with FlhF to prevent its polar localization, and thereby negatively regulates the number of flagella.

Figure 3

FlhF is a signal recognition particle (SRP)-type guanosine triphosphate (GTP)ase. (a) Crystal structure of the FtsY/Ffh heterodimer from Thermus aquaticus (PDB ID 1RJ9 [37]). The non-hydrolyzable guanosine triphosphate (GTP) analog β,γ-methyleneguanosine 5′-triphosphate (GMPPCP) stabilizes the heterodimer, and complex formation aligns the two molecules of this GTP analog in the composite active site. (b) Domain structure of FlhF proteins. Bacillus subtilis FlhF consists of 366 amino acids (41 kDa) with a smaller B domain than Vibrio alginolyticus FlhF (505 amino acids, 57 kDa). FlhF is composed of the function-unknown B domain, the regulatory N domain, and the G domain that contains the GTPase motif (I-IV). (c) Crystal structure of the NG domain homodimer from Bacillus subtilis FlhF in complex with the peptide containing N-terminal 23 residues of FlhG (PDB ID 3SYN [40]). FlhF is shown in light blue, and the FlhG peptide is shown in green. Guanosine diphosphate (GDP) and aluminum fluoride are shown as stick representations, and Mg²⁺ ions are shown in dark green. Residues mutated in corresponding V. alginolyticus proteins are highlighted by blue (function reduced) or red (abolished) balls with the residue number of Vibrio protein. The putative catalytic site (R334 of Vibrio FlhF) is also indicated. For simplicity, the above residues are highlighted only in one protomer. In Vibrio FlhF, alanine substitution of the catalytic residue (R334A) did not affect its function, indicating that GTP binding, but not hydrolysis, is essential.

Figure 4

Proposed model for the regulation of Vibrio alginolyticus polar flagellar transcription hierarchy. This model is based on reports for Vibrio cholerae and Vibrio parahaemolyticus [54,55], whose flagellar genes are highly similar. The master regulator FlaK, which belongs to class 1 as a sole member, regulates downstream flagellar genes. FlaK activity is negatively regulated by FlhG, or FlhG may inhibit the transcription of flaK. The signaling molecule c-di-GMP also negatively regulates FlaK activity.

Figure 5

HubP, the third factor that regulates the number of polar flagella in Vibrio alginolyticus. (a) Domain structure of V. alginolyticus HubP, a single transmembrane protein of 1444 amino acids (≈159 kDa) with a large cytoplasmic region. The LysM domain in the N-terminal periplasmic region functions in anchoring HubP to the peptidoglycan layer. The cytoplasmic repeat sequence and its repetition numbers varies among Vibrio species. (b) HubP functions as the polar “hub”. HubP localizes at the cell pole and anchors three ParA-like proteins at its large cytoplasmic platform. FlhF has an intrinsic property to localize at the cell pole, but FlhG polar localization is dependent on HubP. (c) Electron micrograph of a NMB303 cell, the hubP deletion strain of V. alginolyticus. It generates multiple sheathed flagella at the cell pole.

Figure 6

FlhG is a MinD/ParA-like ATPase that negatively regulates polar flagellar number. (a) Domain structures of Escherichia coli MinD (Ec MinD) and V. alginolyticus FlhG (Va FlhG). FlhG has a slightly longer N-terminal region than MinD, which functions in the stimulation of FlhF GTPase activity. (b) Crystal structures of E. coli MinD dimer in complex with ATP (PDB ID: 3Q9L [62]) and Geobacillus thermodenitrificans FlhG dimer in complex with adenosine diphosphate (ADP) (PDB ID: 4RZ3 [60]). ATP and ADP are shown by stick representations, and important conserved residues are colored red. (c) Model for the regulation of the number of polar flagella in Vibrio alginolyticus. GDP-bound FlhF and ADP-bound FlhG are in an inactive state, interact with each other and remain in the cytoplasm. When GTP is bound, FlhF becomes active and localizes at the cell pole to facilitate flagellation. Likewise, the ATP-bound active form of FlhG localizes to the cell pole via the landmark membrane protein HubP to inhibit FlhF activity. The inhibition of FlhF polar localization (1) and its activity (2) by FlhG optimizes the number of flagella into becoming a single one. (d) Working models to explain nonflagellated or hyperflagellated phenotypes of FlhG or HubP mutants of V. alginolyticus. FlhG D171A, a putative activated mutant, inhibits polar flagellation by more localization of FlhG at the cell pole. FlhG K31A, a nonfunctional mutant, causes hyperflagellation because it cannot localize at the cell pole. K31A also cannot inhibit FlaK so that more flagellar proteins are synthesized. The ΔhubP strain produces the wild-type level of flagellar proteins, but its polar flagellar number increases because FlhG cannot localize at the cell pole.

Figure 7

SflA represses lateral flagellation in the absence of both FlhF and FlhG in Vibrio alginolyticus. (a) Electron micrograph of a LPN4 cell, the ΔflhFGΔsflA mutant. The sheathed flagella were formed at lateral positions, as indicated by the white arrows. (b) Domain structure of SflA. SflA is synthesized as a precursor with an N-terminal signal sequence that is cleaved during maturation (the cleavage site is shown as a black arrowhead). (c) Model of the molecular architecture of the SflA dimer. Unknown binding partner proteins bind to the concave surface of the N-terminal tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) domain of SflA, as indicated by the broken arrows. The DnaJ domain is activated by the binding signal transmitted through the membrane, and interacts with an unknown partner protein to suppress the formation of the sheathed flagellum at peritrichous cell surfaces or promote it at the cell pole. IM, inner membrane.

Figure 8

Model for the regulation of the biogenesis of a single polar flagellum in Vibrio alginolyticus. Five regulatory mechanisms are involved in this proposed model. When flagellar components are synthesized and accumulate, FlhG negatively acts on the master regulator FlaK to downregulate the expression of polar flagellar genes (1). FlhF forms a homodimer in complex with GTP and localizes at the cell pole. FlhF facilitates the accumulation of the MS-ring protein FliF at the cell pole and thereby promotes the MS-ring formation (2). Inactive FlhF and FlhG interact with each other and remain in the cytoplasm (3), and GTP-bound FlhG is activated by HubP at the cell pole and negatively acts on FlhF to inactivate the FlhF dimer at the cell pole (4). In addition to the transcriptional regulation, these post-translational mechanisms optimize polar FlhF activity that allows the cell to generate only a single polar flagellum. Meanwhile, SflA inhibits sheathed flagellar formation at lateral positions by negatively acting on its assembly step (5). FlhF activity is dominant over SflA at the cell pole, so that the effect of SflA, localized at the cell pole via HubP, is suppressed.