A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for ‘Nanoarchaeum equitans’, the only cultivated member of the ‘Nanoarchaeota’. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/IT=DSM 18386T=JCM 14125T).

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two diVerent electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains Wbres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and diVerent to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the midplane. Their cytoplasm exhibits two types of Wbres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin Wbres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.Communicated by Erko Stackebrandt. Electronic supplementary materialThe online version of this article

SummaryThe membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113°C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an a-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement.

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

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