Dataset Information

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions.

ABSTRACT: The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium.Despite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled.

SUBMITTER: Osorio-Valeriano M

PROVIDER: S-EPMC4719438 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions.

Osorio-Valeriano Manuel M de la Mora Javier J Camarena Laura L Dreyfus Georges G

Journal of bacteriology 20151116 3

<h4>Unlabelled</h4>The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, an ...[more]

PMID: 26574514

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Project description:In this work, we have characterized the soluble lytic transglycosylase (SltF) from Rhodobacter sphaeroides that interacts with the scaffolding protein FlgJ in the periplasm to open space at the cell wall peptidoglycan heteropolymer for the emerging rod. The characterization of the genetic context of flgJ and sltF in alphaproteobacteria shows that these two separate genes coexist frequently in a flagellar gene cluster. Two domains of unknown function in SltF were studied, and the results show that the deletion of a 17-amino-acid segment near the N terminus does not show a recognizable phenotype, whereas the deletion of 47 and 95 amino acids of the C terminus of SltF disrupts the interaction with FlgJ without affecting the transglycosylase catalytic activity of SltF. These mutant proteins are unable to support swimming, indicating that the physical interaction between SltF and FlgJ is central for flagellar formation. In a maximum likelihood tree of representative lytic transglycosylases, all of the flagellar SltF proteins cluster in subfamily 1F. From this analysis, it was also revealed that the lytic transglycosylases related to the type III secretion systems present in pathogens cluster with the closely related flagellar transglycosylases.IMPORTANCE Flagellar biogenesis is a highly orchestrated event where the flagellar structure spans the bacterial cell envelope. The rod diameter of approximately 4 nm is larger than the estimated pore size of the peptidoglycan layer; hence, its insertion requires the localized and controlled lysis of the cell wall. We found that a 47-residue domain of the C terminus of the lytic transglycosylase (LT) SltF of R. sphaeroides is involved in the recognition of the rod chaperone FlgJ. We also found that in many alphaproteobacteria, the flagellar cluster includes a homolog of SltF and FlgJ, indicating that association of an LT with the flagellar machinery is ancestral. A maximum likelihood tree shows that family 1 of LTs segregates into seven subfamilies.

Project description:The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.

Project description:UnlabelledRhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 μm and a diameter of 1.4 μm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional.ImportanceR. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.

Dataset Information

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions.

Publications

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions.

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