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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.


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Transcripts observed for the P. furiosus flagellar operon. (A) Flagellar operon of Pyrococcus furiosus with neighboring genes in the upper part. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. Arrows in the lower part indicate cotranscripts identified via RT-PCR. (B) PCR data using genomic DNA as positive control; forward primer was Pfu-flaB0_f, reverse primers were Pfu-flaB0_r to Pfu-flaJ_r. (C) PCR data using cDNA after reverse transcription of isolated RNA and the same primers as given in (B); (data for all other transcripts are found in Supplementary Figure S1). (D) Northern blot experiments using a flaB0 probe. RNA was isolated from late exponentially growing cells (lane 1) and cells in stationary phase (lane 2) and separated in a denaturing agarose gel. The gel migration behavior of an RNA standard is indicated to the left.
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Figure 6: Transcripts observed for the P. furiosus flagellar operon. (A) Flagellar operon of Pyrococcus furiosus with neighboring genes in the upper part. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. Arrows in the lower part indicate cotranscripts identified via RT-PCR. (B) PCR data using genomic DNA as positive control; forward primer was Pfu-flaB0_f, reverse primers were Pfu-flaB0_r to Pfu-flaJ_r. (C) PCR data using cDNA after reverse transcription of isolated RNA and the same primers as given in (B); (data for all other transcripts are found in Supplementary Figure S1). (D) Northern blot experiments using a flaB0 probe. RNA was isolated from late exponentially growing cells (lane 1) and cells in stationary phase (lane 2) and separated in a denaturing agarose gel. The gel migration behavior of an RNA standard is indicated to the left.

Mentions: We detected different length cotranscripts for each of the genes of the flagellar operon with exception of flaJ were only the single gene transcript was found (Figure 6A). The original data using the different primers are shown exemplarily for flaB0 in Figures 6B,C, all other data are given in Supplementary Figure S1. Several transcripts including flaB0 were found whereof the largest with ca. 3.1 kb contained all three flagellins, flaC, and flaD. Besides, various transcripts for the genes flaF-flaI were detected. Interestingly, we found a transcript containing hth and fam whereas flaJ and PF0329 were never part of a cotranscript. Analyses of RNA of cells from different growth phases showed that the transcripts changed over time; the original data are shown exemplarily in Figure 7. In early exponential phase, only few short transcripts were present compared to late exponential and stationary phase indicating that the flagellar operon is transcribed only to a limited degree in early exponential growth phase.


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)

Transcripts observed for the P. furiosus flagellar operon. (A) Flagellar operon of Pyrococcus furiosus with neighboring genes in the upper part. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. Arrows in the lower part indicate cotranscripts identified via RT-PCR. (B) PCR data using genomic DNA as positive control; forward primer was Pfu-flaB0_f, reverse primers were Pfu-flaB0_r to Pfu-flaJ_r. (C) PCR data using cDNA after reverse transcription of isolated RNA and the same primers as given in (B); (data for all other transcripts are found in Supplementary Figure S1). (D) Northern blot experiments using a flaB0 probe. RNA was isolated from late exponentially growing cells (lane 1) and cells in stationary phase (lane 2) and separated in a denaturing agarose gel. The gel migration behavior of an RNA standard is indicated to the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Transcripts observed for the P. furiosus flagellar operon. (A) Flagellar operon of Pyrococcus furiosus with neighboring genes in the upper part. All genes are transcribed from the negatively oriented DNA strand, but are shown here from left to right for easier orientation. Arrows in the lower part indicate cotranscripts identified via RT-PCR. (B) PCR data using genomic DNA as positive control; forward primer was Pfu-flaB0_f, reverse primers were Pfu-flaB0_r to Pfu-flaJ_r. (C) PCR data using cDNA after reverse transcription of isolated RNA and the same primers as given in (B); (data for all other transcripts are found in Supplementary Figure S1). (D) Northern blot experiments using a flaB0 probe. RNA was isolated from late exponentially growing cells (lane 1) and cells in stationary phase (lane 2) and separated in a denaturing agarose gel. The gel migration behavior of an RNA standard is indicated to the left.
Mentions: We detected different length cotranscripts for each of the genes of the flagellar operon with exception of flaJ were only the single gene transcript was found (Figure 6A). The original data using the different primers are shown exemplarily for flaB0 in Figures 6B,C, all other data are given in Supplementary Figure S1. Several transcripts including flaB0 were found whereof the largest with ca. 3.1 kb contained all three flagellins, flaC, and flaD. Besides, various transcripts for the genes flaF-flaI were detected. Interestingly, we found a transcript containing hth and fam whereas flaJ and PF0329 were never part of a cotranscript. Analyses of RNA of cells from different growth phases showed that the transcripts changed over time; the original data are shown exemplarily in Figure 7. In early exponential phase, only few short transcripts were present compared to late exponential and stationary phase indicating that the flagellar operon is transcribed only to a limited degree in early exponential growth phase.

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