Project description:Spirochetes are long, thin, motile, helical or flat wave bacteria that are unique among the prokaryotes by having flagella or axial filaments confined to an internal periplasmic space. These flagella are complex organelles that can play major roles in bacterial pathogenicity and are used as propellers allowing bacteria to move through liquids, viscous environments or along surfaces. While most bacteria species use transcriptional regulatory cascades to regulate the synthesis and assembly of their flagella, spirochetes employ an unusual post-transcriptional mechanism. Using next generation sequencing, we characterized a natural mutant of the relapsing fever spirochete Borrelia hermsii lacking the flagellar export apparatus protein FliH. The mutant was non-motile, uncoiled and straight compared to the wild-type spirochetes. The B. hermsii fliH mutant produced only a residual amount of the major flagellin protein FlaB, which was correlated with a reduced number of periplasmic flagella. The amounts of flaB transcript were comparable in the fliH mutant and the wild-type strain. The synthesis of FlaB, the motility and the infectivity of the fliH mutant were rescued by trans-complementation. This report reveals a new function for FliH, and we propose that this regulator of the flagellar export apparatus influences the post-transcriptional processing of the flagella, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their flat-wave morphology and motility. While other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ posttranscriptional mechanism(s) that are only partially known. In the present study, we characterize a non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcriptome analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major flagellar filament core protein FlaB. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete while the amount of FlaA was similar to the wild-type level. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the posttranscriptional level by influencing the synthesis of FlaB.

Project description:Spirochetes are long, thin, motile, helical or flat wave bacteria that are unique among the prokaryotes by having flagella or axial filaments confined to an internal periplasmic space. These flagella are complex organelles that can play major roles in bacterial pathogenicity and are used as propellers allowing bacteria to move through liquids, viscous environments or along surfaces. While most bacteria species use transcriptional regulatory cascades to regulate the synthesis and assembly of their flagella, spirochetes employ an unusual post-transcriptional mechanism. Using next generation sequencing, we characterized a natural mutant of the relapsing fever spirochete Borrelia hermsii lacking the flagellar export apparatus protein FliH. The mutant was non-motile, uncoiled and straight compared to the wild-type spirochetes. The B. hermsii fliH mutant produced only a residual amount of the major flagellin protein FlaB, which was correlated with a reduced number of periplasmic flagella. The amounts of flaB transcript were comparable in the fliH mutant and the wild-type strain. The synthesis of FlaB, the motility and the infectivity of the fliH mutant were rescued by trans-complementation. This report reveals a new function for FliH, and we propose that this regulator of the flagellar export apparatus influences the post-transcriptional processing of the flagella, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their flat-wave morphology and motility. While other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ posttranscriptional mechanism(s) that are only partially known. In the present study, we characterize a non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcriptome analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major flagellar filament core protein FlaB. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete while the amount of FlaA was similar to the wild-type level. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the posttranscriptional level by influencing the synthesis of FlaB. Borrelia hermsii wild type vs. motility mutant

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

Periplasmic flagellar export apparatus protein, FliH, is involved in post-transcriptional regulation of FlaB, motility and virulence of the relapsing fever spirochete Borrelia hermsii.

Publications

Periplasmic flagellar export apparatus protein, FliH, is involved in post-transcriptional regulation of FlaB, motility and virulence of the relapsing fever spirochete Borrelia hermsii.

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