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Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.

ABSTRACT: The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported herein is the quantitative characterization of interactions between the cytoplasmic domain of FlhB (FlhBC) and other export apparatus proteins including FliK, FlhAC and FliI. FliK and FlhAC bound with micromolar affinity. KD for FliI binding in the absence of ATP was 84 nM. ATP-induced oligomerization of FliI induced kinetic changes, stimulating fast-on, fast-off binding and lowering affinity. Full length FlhB purified under solubilizing, nondenaturing conditions formed a stable dimer via its transmembrane domain and stably bound FliH. Together, the present results support the previously hypothesized central role of FlhB and elucidate the dynamics of protein-protein interactions in type III secretion.

SUBMITTER: McMurry JL

PROVIDER: S-EPMC4526367 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.

McMurry Jonathan L JL Minamino Tohru T Furukawa Yukio Y Francis Joshua W JW Hill Stephanie A SA Helms Katy A KA Namba Keiichi K

PloS one 20150805 8

The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported ...[more]

PMID: 26244937

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Project description:The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.

Project description:FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhAC and FlhBC) coordinate flagellar protein export with assembly. FlhBC undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhBCN and FlhBCC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55?nm in Salmonella As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. The mechanism of export switching remains unclear. Here, we report the role of FlhBC cleavage in the switching mechanism. Photo-cross-linking experiments revealed that the flhB(N269A) and flhB(P270A) mutations did not affect the binding affinity of FlhBC for FliK. Genetic analysis of the flhB(P270A) mutant revealed that the P270A mutation affects a FliK-dependent conformational change of FlhBC, thereby inhibiting the substrate specificity switching. The flhA(A489E) mutation in FlhAC suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhBC and FlhAC is critical for the export switching. We propose that the interaction between FliKC and a cleaved form of FlhBC promotes a conformational change in FlhBC responsible for the termination of hook-type protein export and a structural remodeling of the FlhAC ring responsible for the initiation of filament-type protein export.IMPORTANCE The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.

Dataset Information

Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.

Publications

Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.

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