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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

The flagellar operon of Pyrococcus furiosus. (A) The revised flagellar operon with neighboring genes. The missing genomic sequence and the flaB0 gene are marked in green. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. (B) Functional predictions of the genes shown in (A).
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Figure 1: The flagellar operon of Pyrococcus furiosus. (A) The revised flagellar operon with neighboring genes. The missing genomic sequence and the flaB0 gene are marked in green. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. (B) Functional predictions of the genes shown in (A).

Mentions: Purification of flagella via isopycnic CsCl-gradient centrifugation and analysis by SDS-PAGE identified one major glycoprotein, whose N-terminal amino acid sequence was determined to be AVGIGTLIVF (Näther et al., 2006). Sequence alignments illustrated that this N-terminal sequence did not perfectly match to the annotated flagellins of P. furiosus or to any of the other proteins translated from the published genome (Robb et al., 2001). More precisely, the N-terminus of protein FlaB2 should read AIGIGTLIVF, but Edman degradation of the major flagellin never indicates any heterogeneity at position 2. In case of protein FlaB1 we found that the published sequence lacks the motif AIGIGTLIVFIAM, which is very highly conserved in all flagellins annotated in the publically available genomes of the genus Pyrococcus. However, this motif is encoded directly in front of the annotated flaB1 gene but misses an upstream in-frame start codon. Based on these findings we decided to resequence the genome region that codes for the flagellins. Indeed, we identified a major mistake in that part of the published P. furiosus genome sequence: a total of 771 bp are missing. By combining this new sequence with the published genome (Robb et al., 2001), the sequence of the flaB1 gene now contains a proper start codon and its N-terminus becomes highly similar to other flagellins. In addition, we detected another ORF coding for a third flagellin which we call flaB0 to keep in line with the existing nomenclature. As a consequence, the flagellar operon of P. furiosus was revised (Figure 1). The missing genomic sequence containing the annotation of the flaB0 gene/FlaB0 protein was submitted to NCBI BankIt; the corresponding GenBank number is KM892551.


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)

The flagellar operon of Pyrococcus furiosus. (A) The revised flagellar operon with neighboring genes. The missing genomic sequence and the flaB0 gene are marked in green. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. (B) Functional predictions of the genes shown in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The flagellar operon of Pyrococcus furiosus. (A) The revised flagellar operon with neighboring genes. The missing genomic sequence and the flaB0 gene are marked in green. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. (B) Functional predictions of the genes shown in (A).
Mentions: Purification of flagella via isopycnic CsCl-gradient centrifugation and analysis by SDS-PAGE identified one major glycoprotein, whose N-terminal amino acid sequence was determined to be AVGIGTLIVF (Näther et al., 2006). Sequence alignments illustrated that this N-terminal sequence did not perfectly match to the annotated flagellins of P. furiosus or to any of the other proteins translated from the published genome (Robb et al., 2001). More precisely, the N-terminus of protein FlaB2 should read AIGIGTLIVF, but Edman degradation of the major flagellin never indicates any heterogeneity at position 2. In case of protein FlaB1 we found that the published sequence lacks the motif AIGIGTLIVFIAM, which is very highly conserved in all flagellins annotated in the publically available genomes of the genus Pyrococcus. However, this motif is encoded directly in front of the annotated flaB1 gene but misses an upstream in-frame start codon. Based on these findings we decided to resequence the genome region that codes for the flagellins. Indeed, we identified a major mistake in that part of the published P. furiosus genome sequence: a total of 771 bp are missing. By combining this new sequence with the published genome (Robb et al., 2001), the sequence of the flaB1 gene now contains a proper start codon and its N-terminus becomes highly similar to other flagellins. In addition, we detected another ORF coding for a third flagellin which we call flaB0 to keep in line with the existing nomenclature. As a consequence, the flagellar operon of P. furiosus was revised (Figure 1). The missing genomic sequence containing the annotation of the flaB0 gene/FlaB0 protein was submitted to NCBI BankIt; the corresponding GenBank number is KM892551.

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