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Crystallization and preliminary X-ray analysis of the FliH-FliI complex responsible for bacterial flagellar type III protein export.

ABSTRACT: The bacterial flagellar proteins are translocated into the central channel of the flagellum by a specific protein-export apparatus for self-assembly at the distal growing end. FliH and FliI are soluble components of the export apparatus and form an FliH2-FliI heterotrimer in the cytoplasm. FliI is an ATPase and the FliH2-FliI complex delivers export substrates from the cytoplasm to an export gate made up of six integral membrane proteins of the export apparatus. In this study, an FliHC fragment consisting of residues 99-235 was co-purified with FliI and the FliHC2-FliI complex was crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 400 as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=133.7, b=147.3, c=164.2?Å, and diffracted to 3.0?Å resolution.

SUBMITTER: Uchida Y

PROVIDER: S-EPMC3515370 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray analysis of the FliH-FliI complex responsible for bacterial flagellar type III protein export.

Uchida Yumiko Y Minamino Tohru T Namba Keiichi K Imada Katsumi K

Acta crystallographica. Section F, Structural biology and crystallization communications 20121030 Pt 11

The bacterial flagellar proteins are translocated into the central channel of the flagellum by a specific protein-export apparatus for self-assembly at the distal growing end. FliH and FliI are soluble components of the export apparatus and form an FliH2-FliI heterotrimer in the cytoplasm. FliI is an ATPase and the FliH2-FliI complex delivers export substrates from the cytoplasm to an export gate made up of six integral membrane proteins of the export apparatus. In this study, an FliHC fragment ...[more]

PMID: 23143238

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Project description:UNLABELLED:The Lyme disease spirochete Borrelia burgdorferi migrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI in B. burgdorferi have not been reported. In this study, fliH and fliI transposon mutants were utilized to dissect the mechanism of the Borrelia type III secretion system. The fliH and fliI mutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants in fliH and fliI were incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of either fliH or fliI resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation of fliH and fliI mutants in trans restored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of either fliH or fliI in B. burgdorferi results in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins. IMPORTANCE:Many bacteria are able to rapidly transport themselves through their surroundings using specialized organelles called flagella. In spiral-shaped organisms called spirochetes, flagella act like inboard motors and give the bacteria the ability to bore their way through dense materials (such as human tissue) in a corkscrew manner. In this article, we studied how two proteins, called FliH and FliI, are important for the production of full-length flagella in the Lyme disease spirochete Borrelia burgdorferi. Mutants with defective production of FliH and FliI have reduced flagellar length and motility; this deficiency in turn affects many aspects of B. burgdorferi's biology, including the ability to undergo cell division and cause disease in mammals. Using a microscopic computed tomography (CT) scan approach called cryo-electron tomography, the structure that contains FliH and FliI was defined in the context of the flagellar motor, providing clues regarding how this amazing nanomachine is assembled and functions.

Dataset Information

Crystallization and preliminary X-ray analysis of the FliH-FliI complex responsible for bacterial flagellar type III protein export.

Publications

Crystallization and preliminary X-ray analysis of the FliH-FliI complex responsible for bacterial flagellar type III protein export.

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