No abstract

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

For almost 200 years scientists have been fascinated by the ornate cell walls of the diatoms. These structures are made of amorphous silica, exhibiting species-specific, mostly porous patterns in the nano-to micrometer range. Recently, from the diatom Cylindrotheca fusiformis unusual phosphoproteins (termed silaffins) and long chain polyamines have been identified and implicated in biosilica formation. However, analysis of the role of silaffins in morphogenesis of species-specific silica structures has so far been hampered by the difficulty of obtaining structural data from these extremely complex proteins. In the present study, the five major silaffins from the diatom Thalassiosira pseudonana (tpSil1H, -1L, -2H, -2L, and -3) have been isolated, functionally analyzed, and structurally characterized, making use of the recently available genome data from this organism. Surprisingly, the silaffins of T. pseudonana and C. fusiformis share no sequence homology but are similar regarding amino acid composition and posttranslational modifications. Silaffins tpSil1H and -2H are higher molecular mass isoforms of tpSil1L and -2L, respectively, generated in vivo by alternative processing of the same precursor polypeptides. Interestingly, only tpSil1H and -2H but not tpSil1L and -2L induce the formation of porous silica patterns in vitro, suggesting that the alternative processing event is an important step in morphogenesis of T. pseudonana biosilica.During evolution many organisms (e.g. diatoms, sponges, radiolaria) have acquired the ability to use the ubiquitous monosilicic acid Si(OH) 4 for the formation of species specifically structured, silica-based exo-or endoskeletons (1). This interesting biomineralization phenomenon is mediated by cellular organic (macro-) molecules that accelerate silicic acid polycondensation and control morphogenesis of the forming silica (2). Diatoms are an extremely large group (Ͼ10,000 species) of unicellular eukaryotic algae that play a major role in biological silica cycling. Within the last few years diatom biosilica-associated proteins (termed silaffins) and long chain polyamines (LCPA) 1 have been identified and hypothesized to represent key components of the diatom biosilica-forming machinery. Silaffins and LCPA exhibit the remarkable ability to induce rapid silica deposition in vitro and to control the nanostructure of the forming silica (3). Therefore, unraveling the correlations between chemical structures, physical properties, and silica-forming activities of silaffins and LCPA will be important for understanding the molecular mechanism of species-specific biosilica nanopatterning. So far, silaffins have only been characterized from the diatom Cylindrotheca fusiformis. They are highly modified proteins/peptides rich in hydroxyamino acids (serine, threonine, hydroxyproline) and lysine residues. Silaffins natSil1A and -1B are O-phosphorylated at numerous sites and contain polyamine-modified lysine residues, features that enable these peptides to rapidly form silica nanospheres in vitr...

Diatoms are single-celled algae that produce intricately structured cell walls made of nanopatterned silica (SiO(2)). The cell wall structure is a species-specific characteristic demonstrating that diatom silica morphogenesis is genetically encoded. Understanding the molecular mechanisms by which a single cell executes the morphogenetic program for the formation of an inorganic material (biomineralization) is not only a fascinating biological problem, but also of great interest for nanomaterials science and technology. Recently, analysis of the organic components associated with diatom silica, the development of techniques for molecular genetic manipulation of diatoms, and two diatom genome sequencing projects are providing insight into the composition and mechanism of the remarkable biosilica-forming machinery.

The biological formation of inorganic materials with complex form (biominerals) is a widespread phenomenon in nature, yet the molecular mechanisms underlying biomineral morphogenesis are not well understood. Among the most fascinating examples of biomineral structures are the intricately patterned, silicified cell walls of diatoms, which contain tightly associated organic macromolecules. From diatom biosilica a highly polyanionic phosphoprotein, termed native silaffin-2 (natSil-2), was isolated that carries unconventional amino acid modifications. natSil-2 lacked intrinsic silica formation activity but was able to regulate the activities of the previously characterized silica-forming biomolecules natSil-1A and long-chain polyamines. Combining natSil-2 and natSil-1A (or long-chain polyamines) generated an organic matrix that mediated precipitation of porous silica within minutes after the addition of silicic acid. Remarkably, the precipitate displayed pore sizes in the range 100 -1000 nm, which is characteristic for diatom biosilica nanopatterns.T he biological formation of intracellular or extracellular skeletons made of amorphous hydrated SiO 2 (biosilica) is relatively frequent among the protists and also occurs in many higher plants (1, 2). To date the organic molecules involved in biosilica formation have mainly been studied in sponges and diatoms (3). From the silica spicules of the sponge Thetya aurantia, proteaselike proteins termed silicateins were characterized that catalyze silica formation from tetraethoxysilane in vitro (4, 5). Diatoms are unicellular algae that have the extraordinary capability to produce an enormous variety of biosilica structures (6). Each diatom species is characterized by a specific biosilica cell wall that contains regularly arranged slits or pores in the size range between 10 and 1,000 nm (nanopatterned biosilica). Biosilica morphogenesis takes place inside the diatom cell within a specialized membrane-bound compartment termed the silica deposition vesicle (SDV). It has been postulated that the SDV contains a matrix of organic macromolecules that not only regulate silica formation but also act as templates to mediate biosilica nanopatterning (7-9). Insight into the nature of this organic matrix has been gained through the characterization of diatom biosilica-associated peptides (silaffins) and long-chain polyamines (LCPA), both of which accelerate silica formation from a silicic acid solution in vitro (10-12). Although native silaffin-1A (natSil-1A) and LCPA, respectively, mediate the formation of unusual silica structures in vitro (networks of irregularly shaped silica bands and large spherical silica particles) (11, 12), none of these are akin to diatom biosilica nanopatterns.Recently, it was shown that phosphorylation of natSil-1A is essential for silica formation activity (12), leading to the speculation that phosphorylated components may be generally important for biosilica morphogenesis. Therefore, a search for additional silica-associated phosphoproteins, which are able...