Molecular analysis of the crenarchaeal flagellum

Abstract

SummaryThe ability to move towards favourable conditions provides fundamental advantages to organisms. Interestingly, flagella as motility structures evolved independently in the bacterial and the archaeal kingdom. Whereas bacterial flagella have been intensively studied, our knowledge regarding the archaeal counterpart is mostly restricted to Euryarchaeota rather than crenarchaeal flagella. We therefore investigated the flagellar assembly system of the crenarchaeal model organism Sulfolobus acidocaldarius in vivo. Promoter studies and qRT-PCR analyses of the flagella gene cluster provided evidence that the expression of the fla genes was induced by tryptone starvation. Moreover, we confirmed presence of a secondary fla promoter within the flaB gene that regulates the transcription of downstream genes flaX-J. Markerless in-frame deletions for all fla genes encoded in the fla gene cluster were constructed. Western blot analysis of all fla deletion strains suggested hierarchical protein interactions during the archaeal flagella assembly. Moreover, functional analysis by thermomicroscopy revealed non-motile cells for each of the mutant strains. Electron micrographs demonstrated that lack of motility coincided with the loss of flagellar assembly. Thus we demonstrated that all seven fla genes are essential for crenarchaeal flagellum assembly and function.

Type IV Pilin Proteins: Versatile Molecular Modules

“…It was recently proposed that these unique structures be called "archaella" to distinguish them from eubacterial flagella (199). Although many components of the archaellar assembly system have been identified by knockout studies, the mechanism by which the system generates torque by use of a pilus-like fiber remains elusive (247). For a comprehensive overview of archaeal flagellin assembly and function, we suggest some recent reviews (151,199).…”

Lrs14 transcriptional regulators influence biofilm formation and cell motility of Crenarchaea

“…TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for total RNA isolation following manufacturer's instructions. The preparation of the complementary DNA and the qRT-PCR were performed as described in Lassak et al, 2012 …”

Agl16, a Thermophilic Glycosyltransferase Mediating the Last Step of N -Glycan Biosynthesis in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

“…Induction and immunostaining of the archaellin FlaB. Pellets of fresh 50-ml cultures of MW001 and the ⌬agl16 strain were resuspended and grown for 4 h at 79°C in 40 ml of Brock medium lacking NZ-amine as a carbon and energy source to induce the production of archaella (33). Centrifuged cells were resolved in 1 ml of 20 mM Tris HCl and 500 mM NaCl buffer.…”

“…7B and C). The analyses were repeated with cell cultures that had undergone tryptone starvation to induce archaellum production, as previously described (33). Even under inducing conditions, archaella could not be detected in either mutant by TEM.…”

“…We reasoned that the reduced FlaB protein concentration in the deletion mutants might be caused by a diminished stability or impaired assembly of the archaellum by the altered N-glycan composition. To test the functionality of the archaellum in the deletion strains, a motility assay was performed as previous described (33). Aliquots of the nonmotile S. acidocaldarius ⌬aapF ⌬flaH deletion strain (33) and the hypermotile S. acidocaldarius ⌬aapF strain (37) were used as control strains.…”