Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone.
ABSTRACT: The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD(4)C(2) complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 A resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination.
Project description:Flagellar biogenesis is controlled by a negative feedback loop. When FliD was secreted at the late step of flagellar assembly, the FliD-FliT complex disassembled and free FliT bound to the FlhDC complex, a master regulator of flagellar biogenesis, subsequently inhibiting the overall expression of flagellar proteins. In this study, we analyzed the role of the FliD C-terminal domain in pentamer formation and interaction with FliT. Our study showed that the FliD L443R mutant exists as a monomer in solution, indicating that the Leu443 residue of FliD, which contributes to its interaction with FliT, plays a crucial role in the pentameric oligomerization of FliD. Consistently, the increased levels of free FliT proteins caused by FliD L443R mutation had negative effects on the gene expression of flagellar synthesis and reduced the expression of flagellar proteins. The lengths of flagella in each cell were significantly reduced in L443R mutant strain, suggesting that normal flagellar biogenesis was impeded. These results suggest that the C-terminal domain of FliD plays a crucial role in the pentameric oligmerization of FliD and the binding of FliT to the C-terminal domain of FliD is critical to inhibit the premature assembly of the FliD pentamer in the cytosol.
Project description:The flagellum is a complex bacterial nanomachine that requires the proper assembly of several different proteins for its function. Dedicated chaperones are central in preventing aggregation or undesired interactions of flagellar proteins, including their targeting to the export gate. FliT is a key flagellar chaperone that binds to several flagellar proteins in the cytoplasm, including its cognate filament-capping protein FliD. We have determined the solution structure of the FliT chaperone in the free state and in complex with FliD and the flagellar ATPase FliI. FliT adopts a four-helix bundle and uses a hydrophobic surface formed by the first three helices to recognize its substrate proteins. We show that the fourth helix constitutes the binding site for FlhA, a membrane protein at the export gate. In the absence of a substrate protein FliT adopts an autoinhibited structure wherein both the binding sites for substrates and FlhA are occluded. Substrate binding to FliT activates the complex for FlhA binding and thus targeting of the chaperone-substrate complex to the export gate. The activation and targeting mechanisms reported for FliT appear to be shared among the other flagellar chaperones.
Project description:Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 A resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the binding properties of FlhA-C. While the interaction of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also keeps FliT associated with FlhA-C and excess of FliT inhibits binding of FliD/FliT, suggesting that empty FliT chaperones stay associated with FliJ after export of FliD. Taken together, these results allow to propose a model that explains how the T3SS may switch from the stoichiometric export of FliD to the high-throughput secretion of flagellin.
Project description:E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage ? and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.
Project description:For self-assembly of the bacterial flagellum, most of the flagellar component proteins synthesized in the cytoplasm are exported by the flagellar type III export apparatus to the growing, distal end. Flagellar protein export is highly organized and well controlled in every step of the flagellar assembly process. Flagellar-specific chaperones not only facilitate the export of their cognate proteins, as well as prevent their premature aggregation in the cytoplasm, but also play a role in fine-tuning flagellar gene expression to be coupled with the flagellar assembly process. FliT is a flagellar-specific chaperone responsible for the export of the filament-capping protein FliD and for negative control of flagellar gene expression by binding to the FlhDC complex. Here we report the crystal structure of Salmonella FliT at 3.2-A resolution. The structural and biochemical analyses clearly reveal that the C-terminal segment of FliT regulates its interactions with the FlhDC complex, FliI ATPase, and FliJ (subunits of the export apparatus), and that its conformational change is responsible for the switch in its binding partners during flagellar protein export.
Project description:During a genetic screen to identify metalloregulated loci in Bacillus subtilis, we isolated a Tn917-lacZ insertion in the second gene of an operon downstream of the flagellin (hag) gene. Sequence analysis indicates that this gene encodes a homolog of the enteric flagellar filament cap protein FliD. The fliD gene is followed by homologs of the fliS and fliT genes. Transcription of the fliD-lacZ fusion is sigma D dependent, with peak expression at the end of logarithmic-phase growth. Like other sigma D-dependent genes, expression of fliD-lacZ is greatly reduced by mutations in genes essential for assembly and function of the basal body and hook complex (class II functions). These results suggest that B. subtilis flagellar genes are organized in a hierarchy of gene expression similar to that found in enteric bacteria with hag and fliD as class III genes. Expression from the fliD operon promoter, but not the hag promoter, is repressed by iron, which suggests that the target of metalloregulation is the promoter rather than the sigma D protein.
Project description:The axial component proteins of the bacterial flagellum are synthesized in the cytoplasm and then translocated into the central channel of the flagellum by the flagellar type III protein-export apparatus for self-assembly at the distal growing end of the flagellum. FliJ is an essential cytoplasmic component of the export apparatus. In this study, Salmonella FliJ with an extra three residues (glycine, serine and histidine) attached to the N-terminus as the remainder of a His tag (GSH-FliJ) was purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 300 as a precipitant. GSH-FliJ crystals grew in the hexagonal space group P6(1)22 or P6(5)22. While the native crystals diffracted to 3.3 A resolution, the diffraction resolution limit of mercury derivatives was extended to 2.1 A. Anomalous and isomorphous difference Patterson maps of the mercury-derivative crystal showed significant peaks in their Harker sections, indicating the usefulness of the derivative data for structure determination.
Project description:The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2(1), with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 A, beta = 112.5 degrees . The crystals diffract X-rays to at least 1.6 A resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 A showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.
Project description:In Salmonella typhimurium, nearly 50 genes are involved in flagellar formation and function and constitute at least 13 different operons. In this study, we examined the transcriptional interaction among the flagellar operons by combined use of Mu d1(Apr Lac) cts62 and Tn10 insertion mutants in the flagellar genes. The results showed that the flagellar operons can be divided into three classes: class I contains only the flhD operon, which is controlled by the cAMP-CAP complex and is required for expression of all of the other flagellar operons; class II contains seven operons, flgA, flgB, flhB, fliA, fliE, fliF, and fliL, which are under control of class I and are required for the expression of class III; class III contains five operons, flgK, fliD fliC, motA, and tar. This ordered cascade of transcription closely parallels the assembly of the flagellar structure. In addition, we found that the fliD defect enhanced expression of the class III operons. This suggests that the fliD gene product may be responsible for repression of the class III operons in the mutants in the class II genes. These results are compared with the cascade model of the flagellar regulon of Escherichia coli proposed previously (Y. Komeda, J. Bacteriol. 170:1575-1581, 1982).
Project description:Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the sigma(28)-FlgM checkpoint, which couples the expression of late flagellar (P(class3)) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (P(class2)) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD(4)C(2)-dependent activator of P(class2)/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD(4)C(2) posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor sigma(28) and the filament-cap chaperone/FlhD(4)C(2) inhibitor FliT. Furthermore, we show that the previously described ability of sigma(28) to activate P(class2)/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping P(class2) and P(class3) promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ's role in swimming and swarming motility.