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

Sequence alignment of the three P. furiosus flagellins. Amino acid identities for the three proteins are indicated by asterisks (*); conservative amino acid exchanges are indicated by colons (:), and semi-conservative amino acid exchanges are indicated by dots (.). The arrow shows the signal peptidase processing site. Bold ladders represent the FlaB1 sequence correction resulting from the resequencing performed in this study. Regions indicated by gray sequences identify the least conserved central part of the proteins used to raise flagellin-specific antibodies (primers used for cloning are given in Materials and Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Sequence alignment of the three P. furiosus flagellins. Amino acid identities for the three proteins are indicated by asterisks (*); conservative amino acid exchanges are indicated by colons (:), and semi-conservative amino acid exchanges are indicated by dots (.). The arrow shows the signal peptidase processing site. Bold ladders represent the FlaB1 sequence correction resulting from the resequencing performed in this study. Regions indicated by gray sequences identify the least conserved central part of the proteins used to raise flagellin-specific antibodies (primers used for cloning are given in Materials and Methods).

Mentions: To ask for the presence of the two minor flagellins in our flagella preparations, we raised specific antibodies against all three flagellins. Because of the highly conserved N- and C-terminal part of FlaB0, FlaB1, and FlaB2, we subcloned the unique central part of each flagellin (gray sequences in Figure 2) and used the peptides for immunization of rabbits. The resulting antisera had a low titer, especially for the FlaB2-peptide. Western blots (data not shown) using these antisera proved that all three flagellins are present in the protein band at around 30 kDa. In addition, purified antibodies were used to immuno-label flagella preparations and cells adherent to carbon-coated gold grids for TEM. Again, we could show that antibodies against sheared P. furiosus flagella detach adherent cells from their solid support as described earlier (Näther et al., 2006). Some single cells, however, remained on the grid and their flagella were clearly labeled over their whole length. In contrast, no signals were detected using any of the antibodies against the recombinant flagellin middle parts. Specific antibodies against FlaB1 and FlaB2 reacted mostly with the ends of purified flagella (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)

Sequence alignment of the three P. furiosus flagellins. Amino acid identities for the three proteins are indicated by asterisks (*); conservative amino acid exchanges are indicated by colons (:), and semi-conservative amino acid exchanges are indicated by dots (.). The arrow shows the signal peptidase processing site. Bold ladders represent the FlaB1 sequence correction resulting from the resequencing performed in this study. Regions indicated by gray sequences identify the least conserved central part of the proteins used to raise flagellin-specific antibodies (primers used for cloning are given in Materials and Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Sequence alignment of the three P. furiosus flagellins. Amino acid identities for the three proteins are indicated by asterisks (*); conservative amino acid exchanges are indicated by colons (:), and semi-conservative amino acid exchanges are indicated by dots (.). The arrow shows the signal peptidase processing site. Bold ladders represent the FlaB1 sequence correction resulting from the resequencing performed in this study. Regions indicated by gray sequences identify the least conserved central part of the proteins used to raise flagellin-specific antibodies (primers used for cloning are given in Materials and Methods).
Mentions: To ask for the presence of the two minor flagellins in our flagella preparations, we raised specific antibodies against all three flagellins. Because of the highly conserved N- and C-terminal part of FlaB0, FlaB1, and FlaB2, we subcloned the unique central part of each flagellin (gray sequences in Figure 2) and used the peptides for immunization of rabbits. The resulting antisera had a low titer, especially for the FlaB2-peptide. Western blots (data not shown) using these antisera proved that all three flagellins are present in the protein band at around 30 kDa. In addition, purified antibodies were used to immuno-label flagella preparations and cells adherent to carbon-coated gold grids for TEM. Again, we could show that antibodies against sheared P. furiosus flagella detach adherent cells from their solid support as described earlier (Näther et al., 2006). Some single cells, however, remained on the grid and their flagella were clearly labeled over their whole length. In contrast, no signals were detected using any of the antibodies against the recombinant flagellin middle parts. Specific antibodies against FlaB1 and FlaB2 reacted mostly with the ends of purified flagella (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