Control of the flagellation pattern in Helicobacter pylori by FlhF and FlhG.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.

OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm are limited. Here, we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa ΔoprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here, we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A ΔoprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective ΔoprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in biofilms.

Structure and Assembly of the Bacterial Flagellum.

The bacterial flagellum is a large macromolecular assembly that acts as propeller, providing motility through the rotation of a long extracellular filament. It is composed of over 20 different proteins, many of them highly oligomeric. Accordingly, it has attracted a huge amount of interest amongst researchers and the wider public alike. Nonetheless, most of its molecular details had long remained elusive.This however has changed recently, with the emergence of cryo-EM to determine the structure of protein assemblies at near-atomic resolution. Within a few years, the atomic details of most of the flagellar components have been elucidated, revealing not only its overall architecture but also the molecular details of its rotation mechanism. However, many questions remained unaddressed, notably on the complexity of the assembly of such an intricate machinery.In this chapter, we review the current state of our understanding of the bacterial flagellum structure, focusing on the recent development from cryo-EM. We also highlight the various elements that still remain to be fully characterized. Finally, we summarize the existing model for flagellum assembly and discuss some of the outstanding questions that are still pending in our understanding of the diversity of assembly pathways.

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation.

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.