Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery of Myxococcus xanthus. PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 of Pseudomonas aeruginosa formed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex.Type IV pili (T4P) 3 are versatile surface structures that are important for various processes, including twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are found on the surfaces of a wide variety of Gram-positive and Gram-negative bacteria and archaea (1-3). T4P systems (T4PS) are related to type II secretion systems (T2SS), which are responsible for the secretion of proteins across the outer membrane of Gram-negative bacteria (4, 5), bacterial competence systems that are involved in the uptake of DNA (6), and systems that are involved in the assembly of archaeal surface structures (7).T4P are highly dynamic structures that undergo cycles of extension and retraction (8, 9). During extensions, the T4P assembly ATPase PilB stimulates the extraction of pilin monomers from the inner membrane (IM) and their incorporation at the base of the pilus fiber. The fiber has a diameter of ϳ6 nm and can extend up to several micrometers in length (10). During retractions, the T4P disassembly ATPase PilT stimulates the removal of pilin monomers from the base of the pilus and their reinsertion into the IM (9, 11). T4P retraction generates forces up to 150 piconewtons (12, 13), pulling a cell forward, and making T4P systems the strongest molecular motors known.The rod-shaped cells of the ␦-proteobacterium Myxococcus xanthus, assemble 5-10 T4P at the leading cell pole that extend and retract to generate cell movemen...

Summary In response to a variety of environmental cues, prokaryotes can switch between a motile and a sessile, biofilm‐forming mode of growth. The regulatory mechanisms and signaling pathways underlying this switch are largely unknown in archaea but involve small winged helix‐turn‐helix DNA‐binding proteins of the archaea‐specific Lrs14 family. Here, we study the Lrs14 member AbfR1 of Sulfolobus acidocaldarius. Small‐angle X‐ray scattering data are presented, which are consistent with a model of dimeric AbfR1 in which dimerization occurs via an antiparallel coiled coil as suggested by homology modeling. Furthermore, solution structure data of AbfR1‐DNA complexes suggest that upon binding DNA, AbfR1 induces deformations in the DNA. The wing residues tyrosine 84 and serine 87, which are phosphorylated in vivo, are crucial to establish stable protein‐DNA contacts and their substitution with a negatively charged glutamate or aspartate residue inhibits formation of a nucleoprotein complex. Furthermore, mutation abrogates the cellular abundance and transcription regulatory function of AbfR1 and thus affects the resulting biofilm and motility phenotype of S. acidocaldarius. This work establishes a novel wHTH DNA‐binding mode for Lrs14‐like proteins and hints at an important role for protein phosphorylation as a signal transduction mechanism for the control of biofilm formation and motility in archaea.

In natural environments microorganisms encounter extreme changes in temperature, pH, osmolarities and nutrient availability. The stress response of many bacterial species has been described in detail, however, knowledge in Archaea is limited. Here, we describe the cellular response triggered by nutrient limitation in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. We measured changes in gene transcription and protein abundance upon nutrient depletion up to 4 h after initiation of nutrient depletion. Transcript levels of 1118 of 2223 protein coding genes and abundance of approximately 500 proteins with functions in almost all cellular processes were affected by nutrient depletion. Our study reveals a significant rerouting of the metabolism with respect to degradation of internal as well as extracellular-bound organic carbon and degradation of proteins. Moreover, changes in membrane lipid composition were observed in order to access alternative sources of energy and to maintain pH homeostasis. At transcript level, the cellular response to nutrient depletion in S. acidocaldarius seems to be controlled by the general transcription factors TFB2 and TFEβ. In addition, ribosome biogenesis is reduced, while an increased protein degradation is accompanied with a loss of protein quality control. This study provides first insights into the early cellular response of Sulfolobus to organic carbon and organic nitrogen depletion.