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

Detection of the flaB0 gene in three different P. furiosus strains. Genomic DNA was isolated from the three strains P. furiosus Vc1T, BBR, and LS and used for PCR amplification with primers 353483f and 351761r. Very clearly a ca. 2.5 kb amplificate was identified in all three strains; if flaB0 would be missing (as in the original sequence) a ca. 1.7 kb amplificate would be expected. Lane 1, strain LS; lane 2, strain BBR; lane 3, strain Vc1T; lane 4, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Detection of the flaB0 gene in three different P. furiosus strains. Genomic DNA was isolated from the three strains P. furiosus Vc1T, BBR, and LS and used for PCR amplification with primers 353483f and 351761r. Very clearly a ca. 2.5 kb amplificate was identified in all three strains; if flaB0 would be missing (as in the original sequence) a ca. 1.7 kb amplificate would be expected. Lane 1, strain LS; lane 2, strain BBR; lane 3, strain Vc1T; lane 4, negative control.

Mentions: We therefore asked if the newly discovered flagellin gene flaB0 is conserved not only in the type strain but also in the two lab derivates. Hence, genomic DNA was isolated and primers 353483f and 351761r were used to amplify the region around flaB0. For all three strains a 2.5 kb fragment was amplified as expected for the presence of flaB0 (Figure 5). As the primer numbers refer to the binding position in the public genome of P. furiosus (Robb et al., 2001), the fragment should be only 1.7 kb in length when no flaB0 would be present. Genomic sequencing of the flaB0 region confirmed the sequence we determined earlier for the missing 771 bp segment in all three strains (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)

Detection of the flaB0 gene in three different P. furiosus strains. Genomic DNA was isolated from the three strains P. furiosus Vc1T, BBR, and LS and used for PCR amplification with primers 353483f and 351761r. Very clearly a ca. 2.5 kb amplificate was identified in all three strains; if flaB0 would be missing (as in the original sequence) a ca. 1.7 kb amplificate would be expected. Lane 1, strain LS; lane 2, strain BBR; lane 3, strain Vc1T; lane 4, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Detection of the flaB0 gene in three different P. furiosus strains. Genomic DNA was isolated from the three strains P. furiosus Vc1T, BBR, and LS and used for PCR amplification with primers 353483f and 351761r. Very clearly a ca. 2.5 kb amplificate was identified in all three strains; if flaB0 would be missing (as in the original sequence) a ca. 1.7 kb amplificate would be expected. Lane 1, strain LS; lane 2, strain BBR; lane 3, strain Vc1T; lane 4, negative control.
Mentions: We therefore asked if the newly discovered flagellin gene flaB0 is conserved not only in the type strain but also in the two lab derivates. Hence, genomic DNA was isolated and primers 353483f and 351761r were used to amplify the region around flaB0. For all three strains a 2.5 kb fragment was amplified as expected for the presence of flaB0 (Figure 5). As the primer numbers refer to the binding position in the public genome of P. furiosus (Robb et al., 2001), the fragment should be only 1.7 kb in length when no flaB0 would be present. Genomic sequencing of the flaB0 region confirmed the sequence we determined earlier for the missing 771 bp segment in all three strains (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