Dynamics of the two stator systems in the flagellar motor of Pseudomonas aeruginosa studied by a bead assay

We developed a robust bead assay for studying flagellar motor behavior of Pseudomonas aeruginosa . Using this assay, we studied the dynamics of the two stator systems in the flagellar motor. We found that the two sets of stators function differently, with MotAB stators providing higher total torque, and MotCD stators ensuring more stable motor speed. The motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to mutants with one set of stators. The bead assay we developed here can be further used to study P. aeruginosa chemotaxis at the level of single cell using the motor behavior as the chemotaxis output. Importance Cells of Pseudomonas aeruginosa possess a single polar flagellum, driven by a rotatory motor powered by two sets of torque-generating units (stators). We developed a robust bead assay for studying the behavior of the flagellar motor in P. aeruginosa , by attaching a microsphere to shortened flagellar filament and using it as an indicator of motor rotation. Using this assay, we revealed the dynamics of the two stator systems in the flagellar motor, and found that the motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to mutants with one set of stators.

Lack of N-terminal segment of the flagellin protein results in the production of a shortened polar flagellum in a deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913

Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellar length and found that a single base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281 , encoding the core segment, and the C-terminal segment, and PSM_A2282 , encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin A2281::82 was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of normal length. Analysis of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicates that, due to the lack of two α-helices, the core of the flagellar filament assembled with A2281 is incomplete, which is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. Importance Polar flagella, which are an essential organelle for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment and a C-terminal segment. Results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913 demonstrate that a single base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament.

On the role of flagella in the adaptation of the PGPR Azospirillum brasilense to life on surfaces

The polar flagellum significantly affects the morphological and behavioral responses of the bacterium Azospirillum brasilense Sp245 to changes in the density of the medium and the maintenance of its biofilms at the interface between solid and liquid media.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense

ABSTRACT Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

Modulation of Flagellar Rotation in Surface-Attached Bacteria: A Circuit for Rapid Surface-Sensing

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. P. aeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in P. aeruginosa, the known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appear to result from previously undescribed effects of cAMP on switch complex proteins and/or the motor-stators associated with them.Author SummaryAttachment to a surface often triggers programs of gene expression that alter the behavior, virulence and fitness of bacteria. Initial contact is usually mediated by surface exposed adhesins, such as flagella or pili/fimbriae, and there is much interest in how these structures might sense and respond to surface attachment. The human bacterial pathogen Pseudomonas aeruginosa usually contacts surfaces via its polar flagellum, the rotary motor that also powers bacterial swimming. We observed that wild-type bacteria quickly stopped rotating their flagellum after surface attachment, but that a mutant lacking the flagellar-associated protein FlhF did not. Using a combination of genetic approaches, we demonstrated that FlhF interacts with a component of the flagellar rotor (FliG) and with a polar scaffolding protein that positively regulates cAMP production (FimV) to stop flagellar rotation and thereby favor bacterial persistence at a surface. We provide evidence that the second messenger cAMP is the likely signal generated by flagellar-mediated surface attachment and show that cAMP is sufficient to alter the behavior of the flagellar motor.

Restoration of polar-flagellum motility and biofilm-forming capacity in the mmsB1 mutant of the alphaproteobacterium Azospirillum brasilense Sp245 points to a new role for a homologue of 3-hydroxyisobutyrate dehydrogenase

The bacterium Azospirillum brasilense can swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella, respectively. They also form biofilms on various interfaces. Experimental data on flagellar assembly and social behaviours in these bacteria are scarce. Here, for the first time, the chromosomal coding sequence mmsB1 for a homologue of 3-hydroxyisobutyrate dehydrogenase (protein accession Nos. ADT80774 and E7CWE2) was shown to play a role in the assembly of motile Fla and in biofilm biomass accumulation. In the previously obtained mutant SK039 of A. brasilense Sp245, an Omegon-Km insertion in mmsB1 was concurrent with changes in cell-surface properties and with suppression of Fla assembly (partial) and Fla-dependent motility (complete). Here, the immotile leaky Fla− mutant SK039 was complemented with the expression vector pRK415-borne mmsB1 gene of Sp245. In the complemented mutant, the elevated relative cell hydrophobicity and changed relative membrane fluidity of SK039 returned to the wild-type levels; also, biofilm biomass accumulation increased and even reached Sp245’s levels under nutritionally rich conditions. In strain SK039 (pRK415-mmsB1), the percentage of cells with Fla became significantly higher than that in mutant SK039, and the Fla-driven swimming velocity was equal to that in strain Sp245.