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Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma.

ABSTRACT: Fibril is a constitutive filament-forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in cell helicity. However, the mechanism driving conformational changes in Fibril remains unknown. We expressed Fibril from S. citri in E. coli for its purification and characterization. Sodium dodecyl sulfate solubilized Fibril filaments and facilitated purification by affinity chromatography. An alternative protocol for obtaining enriched insoluble Fibril filaments was standardized using density gradient centrifugation. Electron microscopy of Fibril purified by these protocols revealed filament bundles. Probable domain boundaries of Fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of α-helical and β-sheet signatures in FT-IR measurements suggests that Fibril filaments consist of an assembly of folded globular domains, and not a β-strand-based aggregation like amyloid fibrils.

SUBMITTER: Harne S

PROVIDER: S-EPMC9489929 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma.

Harne Shrikant S Gayathri Pananghat P

iScience 20220903 10

Fibril is a constitutive filament-forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in cell helicity. However, the mechanism driving conformational changes in Fibril remains unknown. We expressed Fibril from S. citri in E. coli for its purification and characterization. Sodium dodecyl sulfate solub ...[more]

PMID: 36157586

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Project description:BACKGROUND: The genus Spiroplasma contains a group of helical, motile, and wall-less bacteria in the class Mollicutes. Similar to other members of this class, such as the animal-pathogenic Mycoplasma and the plant-pathogenic 'Candidatus Phytoplasma', all characterized Spiroplasma species were found to be associated with eukaryotic hosts. While most of the Spiroplasma species appeared to be harmless commensals of insects, a small number of species have evolved pathogenicity toward various arthropods and plants. In this study, we isolated a novel strain of honeybee-associated S. melliferum and investigated its genetic composition and evolutionary history by whole-genome shotgun sequencing and comparative analysis with other Mollicutes genomes. RESULTS: The whole-genome shotgun sequencing of S. melliferum IPMB4A produced a draft assembly that was ~1.1 Mb in size and covered ~80% of the chromosome. Similar to other Spiroplasma genomes that have been studied to date, we found that this genome contains abundant repetitive sequences that originated from plectrovirus insertions. These phage fragments represented a major obstacle in obtaining a complete genome sequence of Spiroplasma with the current sequencing technology. Comparative analysis of S. melliferum IPMB4A with other Spiroplasma genomes revealed that these phages may have facilitated extensive genome rearrangements in these bacteria and contributed to horizontal gene transfers that led to species-specific adaptation to different eukaryotic hosts. In addition, comparison of gene content with other Mollicutes suggested that the common ancestor of the SEM (Spiroplasma, Entomoplasma, and Mycoplasma) clade may have had a relatively large genome and flexible metabolic capacity; the extremely reduced genomes of present day Mycoplasma and 'Candidatus Phytoplasma' species are likely to be the result of independent gene losses in these lineages. CONCLUSIONS: The findings in this study highlighted the significance of phage insertions and horizontal gene transfer in the evolution of bacterial genomes and acquisition of pathogenicity. Furthermore, the inclusion of Spiroplasma in comparative analysis has improved our understanding of genome evolution in Mollicutes. Future improvements in the taxon sampling of available genome sequences in this group are required to provide further insights into the evolution of these important pathogens of humans, animals, and plants.

Dataset Information

Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma.

Publications

Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium <i>Spiroplasma</i>.

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