Limits...
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

History of the P. furiosus strains used in this study. The type strain Vc1T was deposited within 6 months after its isolation at DSMZ. Strains P. furiosus LS and BBR were regenerated at different times from our in-house culture collection and thereafter repeatedly grown and stored at 4°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263178&req=5

Figure 4: History of the P. furiosus strains used in this study. The type strain Vc1T was deposited within 6 months after its isolation at DSMZ. Strains P. furiosus LS and BBR were regenerated at different times from our in-house culture collection and thereafter repeatedly grown and stored at 4°C.

Mentions: The genome of P. furiosus has been reported to be dynamic (Bridger et al., 2012) — a feature of this hyperthermophile we experienced also in our Regensburg labs. Over the years, we have identified at least 2 strains differing from the original P. furiosus isolate whose origin/history is shown in Figure 4. The original strain named Vc1 was deposited as type strain DSM3638T at the German Culture Collection (Deutsche Sammlung für Mikroorganismen und Zellkulturen, DSMZ) ca. 6 months after its isolation. The same isolate was repeatedly regrown (for ca. 7 years) from stocks stored at 4°C and deposited in 1992 in our Regensburg Culture Collection, (Bakterienbank Regensburg, BBR). Therefrom strain LS was regenerated in 2004 and was repeatedly regrown from stocks stored at 4°C. Another derivate, strain BBR was regenerated from our in-house culture collection in 2008 and repeatedly regrown from stocks stored at 4°C. The three strains of P. furiosus, namely Vc1T, LS, and BBR, differ with respect to their binding behavior to various surfaces if tested as described (Näther et al., 2006), they express different amounts of flagella and their cell morphology differs drastically (Bellack, 2011; data will be described in detail elsewhere).


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)

History of the P. furiosus strains used in this study. The type strain Vc1T was deposited within 6 months after its isolation at DSMZ. Strains P. furiosus LS and BBR were regenerated at different times from our in-house culture collection and thereafter repeatedly grown and stored at 4°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: History of the P. furiosus strains used in this study. The type strain Vc1T was deposited within 6 months after its isolation at DSMZ. Strains P. furiosus LS and BBR were regenerated at different times from our in-house culture collection and thereafter repeatedly grown and stored at 4°C.
Mentions: The genome of P. furiosus has been reported to be dynamic (Bridger et al., 2012) — a feature of this hyperthermophile we experienced also in our Regensburg labs. Over the years, we have identified at least 2 strains differing from the original P. furiosus isolate whose origin/history is shown in Figure 4. The original strain named Vc1 was deposited as type strain DSM3638T at the German Culture Collection (Deutsche Sammlung für Mikroorganismen und Zellkulturen, DSMZ) ca. 6 months after its isolation. The same isolate was repeatedly regrown (for ca. 7 years) from stocks stored at 4°C and deposited in 1992 in our Regensburg Culture Collection, (Bakterienbank Regensburg, BBR). Therefrom strain LS was regenerated in 2004 and was repeatedly regrown from stocks stored at 4°C. Another derivate, strain BBR was regenerated from our in-house culture collection in 2008 and repeatedly regrown from stocks stored at 4°C. The three strains of P. furiosus, namely Vc1T, LS, and BBR, differ with respect to their binding behavior to various surfaces if tested as described (Näther et al., 2006), they express different amounts of flagella and their cell morphology differs drastically (Bellack, 2011; data will be described in detail elsewhere).

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