Functional dissection of the three N-terminal general secretory pathway domains and the Walker motifs of the traffic ATPase PilF from Thermus thermophilus.

The traffic ATPase PilF of Thermus thermophilus powers pilus assembly as well as uptake of DNA. PilF differs from other traffic ATPases by a triplicated general secretory pathway II, protein E, N-terminal domain (GSPIIABC). We investigated the in vivo and in vitro roles of the GSPII domains, the Walker A motif and a catalytic glutamate by analyzing a set of PilF deletion derivatives and pilF mutants.

Molecular insights into lipid-assisted Ca regulation of the TRP channel Polycystin-2.

Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca signaling pathways. Malfunctioning Ca regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations.

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding.

The Thermus thermophilus comEA/comEC operon is associated with DNA binding and regulation of the DNA translocator and type IV pili.

Natural transformation systems and type IV pili are linked in many naturally competent bacteria. In the Gram-negative bacterium Thermus thermophilus, a leading model organism for studies of DNA transporters in thermophilic bacteria, seven competence proteins play a dual role in both systems, whereas two competence genes, comEA and comEC, are suggested to represent unique DNA translocator proteins. Here we show that the T.

Different effects of MglA and MglB on pilus-mediated functions and natural competence in Thermus thermophilus.

The thermophilic bacterium Thermus thermophilus is known for its high natural competence. Uptake of DNA is mediated by a DNA translocator that shares components with type IV pili. Localization and function of type IV pili in other bacteria depend on the cellular localization at the poles of the bacterium, a process that involves MglA and MglB.

Nonacetogenic growth of the acetogen Acetobacterium woodii on 1,2-propanediol.

Acetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO2 in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD.

Zinc and ATP binding of the hexameric AAA-ATPase PilF from Thermus thermophilus: role in complex stability, piliation, adhesion, twitching motility, and natural transformation.

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability.

Environmental factors affecting the expression of type IV pilus genes as well as piliation of Thermus thermophilus.

The thermophilic bacterium Thermus thermophilus HB27 is known for its highly efficient natural transformation system, which has become a model system to study the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter is functionally linked to type IV pili (T4P), which are essential for twitching motility and adhesion to solid surfaces. However, the pilus structures themselves are dispensable for natural transformation.

Type IV pilus biogenesis, twitching motility, and DNA uptake in Thermus thermophilus: discrete roles of antagonistic ATPases PilF, PilT1, and PilT2.

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane.

Cell-free expression and assembly of ATP synthase.

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition.

Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells.

Background: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism.

Improved specimen reconstruction by Hilbert phase contrast tomography.

The low signal-to-noise ratio (SNR) in images of unstained specimens recorded with conventional defocus phase contrast makes it difficult to interpret 3D volumes obtained by electron tomography (ET). The high defocus applied for conventional tilt series generates some phase contrast but leads to an incomplete transfer of object information. For tomography of biological weak-phase objects, optimal image contrast and subsequently an optimized SNR are essential for the reconstruction of details such as macromolecular assemblies at molecular resolution.