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Pyrococcus furiosus flagella: biochemical and transcriptional analyses identify the newly detected flaB0 gene to encode the major flagellin.

Näther-Schindler DJ, Schopf S, Bellack A, Rachel R, Wirth R - Front Microbiol (2014)

Bottom Line: Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments.A total of 771 bp are missing in the data base, resulting in the correction of the previously unusual N-terminal sequence of flagellin FlaB1 and in the identification of a third flagellin.Analysing the RNA of cells from different growth phases, we found that the length and number of detected cotranscript increased over time suggesting that the flagellar operon is transcribed mostly in late exponential and stationary growth phase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Archaea Center, University of Regensburg Regensburg, Germany ; Plant Development, Department of Biology I, Biocenter of the Ludwig Maximilian University of Munich Planegg-Martinsried, Germany.

ABSTRACT
We have described previously that the flagella of the Euryarchaeon Pyrococcus furiosus are multifunctional cell appendages used for swimming, adhesion to surfaces and formation of cell-cell connections. Here, we characterize these organelles with respect to their biochemistry and transcription. Flagella were purified by shearing from cells followed by CsCl-gradient centrifugation and were found to consist mainly of a ca. 30 kDa glycoprotein. Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments. The N-terminal sequence of the main flagellin was determined by Edman degradation, but none of the genes in the complete genome code for a protein with that N-terminus. Therefore, we resequenced the respective region of the genome, thereby discovering that the published genome sequence is not correct. A total of 771 bp are missing in the data base, resulting in the correction of the previously unusual N-terminal sequence of flagellin FlaB1 and in the identification of a third flagellin. To keep in line with the earlier nomenclature we call this flaB0. Very interestingly, the previously not identified flaB0 codes for the major flagellin. Transcriptional analyses of the revised flagellar operon identified various different cotranscripts encoding only a single protein in case of FlaB0 and FlaJ or up to five proteins (FlaB0-FlaD). Analysing the RNA of cells from different growth phases, we found that the length and number of detected cotranscript increased over time suggesting that the flagellar operon is transcribed mostly in late exponential and stationary growth phase.

No MeSH data available.


Related in: MedlinePlus

Repolymerization of denatured flagella. (A) SDS-PAGE: Flagella purified by CsCl-gradient centrifugation were denatured into monomeric flagellins by SDS and heat denaturation (lane 1). After extensive dialysis against 5 mM HEPES buffer only single flagellins were observed (lane 2). The denatured flagellins were used for polymerization assays at: 8°C (lanes 3 and 7), 37°C (lanes 4 and 8), 60°C (lanes 5 and 9), and 90°C (lanes 6 and 10). Analysis was done after 1 (lane 3–6) or 6 days (lane 7–10) of incubation. (B–D) show TEM analyses of: (B) the flagella preparation; (C) denatured flagellins (lane 2); (D) the result from a 90°C repolymerization after 1 day (lane 6). Size bars are 100 nm, each.
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Figure 3: Repolymerization of denatured flagella. (A) SDS-PAGE: Flagella purified by CsCl-gradient centrifugation were denatured into monomeric flagellins by SDS and heat denaturation (lane 1). After extensive dialysis against 5 mM HEPES buffer only single flagellins were observed (lane 2). The denatured flagellins were used for polymerization assays at: 8°C (lanes 3 and 7), 37°C (lanes 4 and 8), 60°C (lanes 5 and 9), and 90°C (lanes 6 and 10). Analysis was done after 1 (lane 3–6) or 6 days (lane 7–10) of incubation. (B–D) show TEM analyses of: (B) the flagella preparation; (C) denatured flagellins (lane 2); (D) the result from a 90°C repolymerization after 1 day (lane 6). Size bars are 100 nm, each.

Mentions: Flagellins from denaturated flagella were incubated at different temperatures to analyze their potential to repolymerize. Incubation for 1 day or longer at temperatures higher than 60°C resulted in aggregation of the ~30 kDa flagellins into high-molecular weight polymers, forming in part also filamentous structures as proven by TEM (Figure 3). Comparing these filaments to native flagella, we found the diameter to be smaller, and no helical ultrastructure was present. Addition of ATP to the samples had no influence on the formation of aggregates or filaments (data not shown).


Pyrococcus furiosus flagella: biochemical and transcriptional analyses identify the newly detected flaB0 gene to encode the major flagellin.

Näther-Schindler DJ, Schopf S, Bellack A, Rachel R, Wirth R - Front Microbiol (2014)

Repolymerization of denatured flagella. (A) SDS-PAGE: Flagella purified by CsCl-gradient centrifugation were denatured into monomeric flagellins by SDS and heat denaturation (lane 1). After extensive dialysis against 5 mM HEPES buffer only single flagellins were observed (lane 2). The denatured flagellins were used for polymerization assays at: 8°C (lanes 3 and 7), 37°C (lanes 4 and 8), 60°C (lanes 5 and 9), and 90°C (lanes 6 and 10). Analysis was done after 1 (lane 3–6) or 6 days (lane 7–10) of incubation. (B–D) show TEM analyses of: (B) the flagella preparation; (C) denatured flagellins (lane 2); (D) the result from a 90°C repolymerization after 1 day (lane 6). Size bars are 100 nm, each.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263178&req=5

Figure 3: Repolymerization of denatured flagella. (A) SDS-PAGE: Flagella purified by CsCl-gradient centrifugation were denatured into monomeric flagellins by SDS and heat denaturation (lane 1). After extensive dialysis against 5 mM HEPES buffer only single flagellins were observed (lane 2). The denatured flagellins were used for polymerization assays at: 8°C (lanes 3 and 7), 37°C (lanes 4 and 8), 60°C (lanes 5 and 9), and 90°C (lanes 6 and 10). Analysis was done after 1 (lane 3–6) or 6 days (lane 7–10) of incubation. (B–D) show TEM analyses of: (B) the flagella preparation; (C) denatured flagellins (lane 2); (D) the result from a 90°C repolymerization after 1 day (lane 6). Size bars are 100 nm, each.
Mentions: Flagellins from denaturated flagella were incubated at different temperatures to analyze their potential to repolymerize. Incubation for 1 day or longer at temperatures higher than 60°C resulted in aggregation of the ~30 kDa flagellins into high-molecular weight polymers, forming in part also filamentous structures as proven by TEM (Figure 3). Comparing these filaments to native flagella, we found the diameter to be smaller, and no helical ultrastructure was present. Addition of ATP to the samples had no influence on the formation of aggregates or filaments (data not shown).

Bottom Line: Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments.A total of 771 bp are missing in the data base, resulting in the correction of the previously unusual N-terminal sequence of flagellin FlaB1 and in the identification of a third flagellin.Analysing the RNA of cells from different growth phases, we found that the length and number of detected cotranscript increased over time suggesting that the flagellar operon is transcribed mostly in late exponential and stationary growth phase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Archaea Center, University of Regensburg Regensburg, Germany ; Plant Development, Department of Biology I, Biocenter of the Ludwig Maximilian University of Munich Planegg-Martinsried, Germany.

ABSTRACT
We have described previously that the flagella of the Euryarchaeon Pyrococcus furiosus are multifunctional cell appendages used for swimming, adhesion to surfaces and formation of cell-cell connections. Here, we characterize these organelles with respect to their biochemistry and transcription. Flagella were purified by shearing from cells followed by CsCl-gradient centrifugation and were found to consist mainly of a ca. 30 kDa glycoprotein. Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments. The N-terminal sequence of the main flagellin was determined by Edman degradation, but none of the genes in the complete genome code for a protein with that N-terminus. Therefore, we resequenced the respective region of the genome, thereby discovering that the published genome sequence is not correct. A total of 771 bp are missing in the data base, resulting in the correction of the previously unusual N-terminal sequence of flagellin FlaB1 and in the identification of a third flagellin. To keep in line with the earlier nomenclature we call this flaB0. Very interestingly, the previously not identified flaB0 codes for the major flagellin. Transcriptional analyses of the revised flagellar operon identified various different cotranscripts encoding only a single protein in case of FlaB0 and FlaJ or up to five proteins (FlaB0-FlaD). Analysing the RNA of cells from different growth phases, we found that the length and number of detected cotranscript increased over time suggesting that the flagellar operon is transcribed mostly in late exponential and stationary growth phase.

No MeSH data available.


Related in: MedlinePlus