DNA-binding and repressor function are prerequisites for the turnover of the tomato heat stress transcription factor HsfB1.

HsfB1 is a central regulator of heat stress (HS) response and functions dually as a transcriptional co-activator of HsfA1a and a general repressor in tomato. HsfB1 is efficiently synthesized during the onset of HS and rapidly removed in the course of attenuation during the recovery phase. Initial results point to a complex regime modulating HsfB1 abundance involving the molecular chaperone Hsp90. However, the molecular determinants affecting HsfB1 stability needed to be established. We provide experimental evidence that DNA-bound HsfB1 is efficiently targeted for degradation when active as a transcriptional repressor. Manipulation of the DNA-binding affinity by mutating the HsfB1 DNA-binding domain directly influences the stability of the transcription factor. During HS, HsfB1 is stabilized, probably due to co-activator complex formation with HsfA1a. The process of HsfB1 degradation involves nuclear localized Hsp90. The molecular determinants of HsfB1 turnover identified in here are so far seemingly unique. A mutational switch of the R/KLFGV repressor motif's arginine and lysine implies that the abundance of other R/KLFGV type Hsfs, if not other transcription factors as well, might be modulated by a comparable mechanism. Thus, we propose a versatile mechanism for strict abundance control of the stress-induced transcription factor HsfB1 for the recovery phase, and this mechanism constitutes a form of transcription factor removal from promoters by degradation inside the nucleus.

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins.

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub-compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.

Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family.

Proteins of the Omp85 family chaperone the membrane insertion of ß-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded ß-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded ß-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc.

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy.

Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a ß-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal ß-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase.

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.

50 years of amino acid hydrophobicity scales: revisiting the capacity for peptide classification.

BACKGROUND: Physicochemical properties are frequently analyzed to characterize protein-sequences of known and unknown function. Especially the hydrophobicity of amino acids is often used for structural prediction or for the detection of membrane associated or embedded ß-sheets and α-helices. For this purpose many scales classifying amino acids according to their physicochemical properties have been defined over the past decades. In parallel, several hydrophobicity parameters have been defined for calculation of peptide properties. We analyzed the performance of separating sequence pools using 98 hydrophobicity scales and five different hydrophobicity parameters, namely the overall hydrophobicity, the hydrophobic moment for detection of the α-helical and ß-sheet membrane segments, the alternating hydrophobicity and the exact ß-strand score. RESULTS: Most of the scales are capable of discriminating between transmembrane α-helices and transmembrane ß-sheets, but assignment of peptides to pools of soluble peptides of different secondary structures is not achieved at the same quality. The separation capacity as measure of the discrimination between different structural elements is best by using the five different hydrophobicity parameters, but addition of the alternating hydrophobicity does not provide a large benefit. An in silico evolutionary approach shows that scales have limitation in separation capacity with a maximal threshold of 0.6 in general. We observed that scales derived from the evolutionary approach performed best in separating the different peptide pools when values for arginine and tyrosine were largely distinct from the value of glutamate. Finally, the separation of secondary structure pools via hydrophobicity can be supported by specific detectable patterns of four amino acids. CONCLUSION: It could be assumed that the quality of separation capacity of a certain scale depends on the spacing of the hydrophobicity value of certain amino acids. Irrespective of the wealth of hydrophobicity scales a scale separating all different kinds of secondary structures or between soluble and transmembrane peptides does not exist reflecting that properties other than hydrophobicity affect secondary structure formation as well. Nevertheless, application of hydrophobicity scales allows distinguishing between peptides with transmembrane α-helices and ß-sheets. Furthermore, the overall separation capacity score of 0.6 using different hydrophobicity parameters could be assisted by pattern search on the protein sequence level for specific peptides with a length of four amino acids.

The evolution of the ribosome biogenesis pathway from a yeast perspective.

Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.

Functional properties of LptA and LptD in Anabaena sp. PCC 7120.

Lipopolysaccharides (LPS) are central components of the outer membrane and consist of Lipid A, the core polysaccharide, and the O-antigen. The synthesis of LPS is initiated at the cytosolic face of the cytoplasmic membrane. The subsequent transport to and across the outer membrane involves multiple lipopolysaccharide transport (Lpt) proteins. Among those proteins, the periplasmic-localized LptA and the outer membrane-embedded LptD participate in the last steps of transfer and insertion of LPS into the outer membrane. While the process is described for proteobacterial model systems, not much is known about the machinery in cyanobacteria. We demonstrate that anaLptD (alr1278) of Anabaena sp. PCC 7120 is important for cell wall function and its pore domain shows a Lipid A sensitive cation-selective gating behavior. The N-terminal domain of anaLptD recognizes anaLptA (alr4067), but not ecLptA. Furthermore, anaLptA specifically interacts with the Lipid A from Anabaena sp. PCC 7120 only, while anaLptD binds to Lipid A isolated from Escherichia coli as well. Based on the comparative analysis of proteins from E. coli and Anabaena sp. we discuss the properties of the cyanobacterial Lpt system.

The outer membrane TolC-like channel HgdD is part of tripartite resistance-nodulation-cell division (RND) efflux systems conferring multiple-drug resistance in the Cyanobacterium Anabaena sp. PCC7120.

The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.

Mdm10 is an ancient eukaryotic porin co-occurring with the ERMES complex.

Mitochondrial ß-barrel proteins fulfill central functions in the outer membrane like metabolite exchange catalyzed by the voltage-dependent anion channel (VDAC) and protein biogenesis by the central components of the preprotein translocase of the outer membrane (Tom40) or of the sorting and assembly machinery (Sam50). The mitochondrial division and morphology protein Mdm10 is another essential outer membrane protein with proposed ß-barrel fold, which has so far only been found in Fungi. Mdm10 is part of the endoplasmic reticulum mitochondria encounter structure (ERMES), which tethers the ER to mitochondria and associates with the SAM complex. In here, we provide evidence that Mdm10 phylogenetically belongs to the VDAC/Tom40 superfamily. Contrary to Tom40 and VDAC, Mdm10 exposes long loops towards both sides of the membrane. Analyses of single loop deletion mutants of Mdm10 in the yeast Saccharomyces cerevisiae reveal that the loops are dispensable for Mdm10 function. Sequences similar to fungal Mdm10 can be found in species from Excavates to Fungi, but neither in Metazoa nor in plants. Strikingly, the presence of Mdm10 coincides with the appearance of the other ERMES components. Mdm10's presence in both unikonts and bikonts indicates an introduction at an early time point in eukaryotic evolution.

The folding capacity of the mature domain of the dual-targeted plant tRNA nucleotidyltransferase influences organelle selection.

tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3'-termini. Therefore this enzymatic activity is found in all cellular compartments, namely the nucleus, cytoplasm, plastids and mitochondria, in which tRNA synthesis or translation occurs. A single gene codes for tRNA-NT in plants, suggesting a complex targeting mechanism. Consistent with this, distinct signals have been proposed for plastidic, mitochondrial and nuclear targeting. Our previous research has shown that in addition to N-terminal targeting information, the mature domain of the protein itself modifies targeting to mitochondria and plastids. This suggests the existence of an as yet unknown determinate for the distribution of dual-targeted proteins between these two organelles. In the present study, we explore the enzymatic and physicochemical properties of tRNA-NT variants to correlate the properties of the enzyme with the intracellular distribution of the protein. We show that alteration of tRNA-NT stability influences its intracellular distribution due to variations in organelle import capacities. Hence the fate of the protein is determined not only by the transit peptide sequence, but also by the physicochemical properties of the mature protein.

Structural and functional analysis of the archaeal endonuclease Nob1.

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.

Chloroplast Omp85 proteins change orientation during evolution.

The majority of outer membrane proteins (OMPs) from gram-negative bacteria and many of mitochondria and chloroplasts are ß-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded ß-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using self-assembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

The influence of fatty acids on the GpA dimer interface by coarse-grained molecular dynamics simulation.

The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA.

The TolC-like protein HgdD of the cyanobacterium Anabaena sp. PCC 7120 is involved in secondary metabolite export and antibiotic resistance.

The role of TolC has largely been explored in proteobacteria, where it functions as a metabolite and protein exporter. In contrast, little research has been carried out on the function of cyanobacterial homologues, and as a consequence, not much is known about the mechanism of cyanobacterial antibiotic uptake and metabolite secretion in general. It has been suggested that the TolC-like homologue of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, termed heterocyst glycolipid deposition protein D (HgdD), is involved in both protein and lipid secretion. To describe its function in secondary metabolite secretion, we established a system to measure the uptake of antibiotics based on the fluorescent molecule ethidium bromide. We analyzed the rate of porin-dependent metabolite uptake and confirmed the functional relation between detoxification and the action of HgdD. Moreover, we identified two major facilitator superfamily proteins that are involved in this process. It appears that anaOmp85 (Alr2269) is not required for insertion or assembly of HgdD, because an alr2269 mutant does not exhibit a phenotype similar to the hgdD mutant. Thus, we could assign components of the metabolite efflux system and describe parameters of detoxification by Anabaena sp. PCC 7120.

Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria.

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.

The protein translocation systems in plants - composition and variability on the example of Solanum lycopersicum.

BACKGROUND: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species. RESULTS: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots. CONCLUSIONS: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum.

Structural elements of the mitochondrial preprotein-conducting channel Tom40 dissolved by bioinformatics and mass spectrometry.

Most mitochondrial proteins are imported into mitochondria from the cytosolic compartment. Proteins destined for the outer or inner membrane, the inter-membrane space, or the matrix are recognized and translocated by the TOM machinery containing the specialized protein import channel Tom40. The latter is a protein with ß-barrel shape, which is suggested to have evolved from a porin-type protein. To obtain structural insights in the absence of a crystal structure the membrane topology of Tom40 from Neurospora crassa was determined by limited proteolysis combined with mass spectrometry. The results were interpreted on the basis of a structural model that has been generated for NcTom40 by using the structure of mouse VDAC-1 as a template and amino acid sequence information of approximately 270 different Tom40 and approximately 480 VDAC amino acid sequences for refinement. The model largely explains the observed accessible cleavage sites and serves as a structural basis for the investigation of physicochemical properties of the ensemble of our Tom40 sequence data set. By this means we discovered two conserved polar slides in the pore interior. One is possibly involved in the positioning of a pore-inserted helix; the other one might be important for mitochondrial pre-sequence peptide binding as it is only present in Tom40 but not in VDAC proteins. The outer surface of the Tom40 barrel reveals two conserved amino acid clusters. They may be involved in binding other components of the TOM complex or bridging components of the TIM machinery of the mitochondrial inner membrane.

The components of the putative iron transport system in the cyanobacterium Anabaena sp. PCC 7120.

Iron uptake in Gram-negative bacteria involves four distinct steps: (i) siderophore synthesis, (ii) siderophore secretion into the extracellular space, (iii) iron chelation by the siderophores, and (iv) siderophore/iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane. This process is well characterized for some proteobacterial systems, but largely unexplored and scarcely investigated in cyanobacteria such as the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Two putative siderophore synthesis clusters have been recently identified in this cyanobacterium. In addition, the export system for the main siderophore, schizokinen, secreted by Anabaena sp. PCC 7120 was described as well as the outer membrane transporter for its import from the extracellular space. We present the identification of components of three additional systems involved in siderophore-mediated iron uptake under iron-limiting conditions, namely TonB3, the ExbB3/ExbD3 and the Fhu systems. The transcription level of these genes is elevated under iron limitations and decreased under excess iron, while the expression levels of other members of these gene families and systems are impacted in distinct ways by other environmental conditions. Mutants of the tonB3, exbB3/exbD3 and fhu genes show an iron starvation phenotype. Thus, Anabaena sp. has a similar, yet distinct system for siderophore-dependent iron uptake compared with other proteobacteria.

Identification of two voltage-dependent anion channel-like protein sequences conserved in Kinetoplastida.

The eukaryotic porin superfamily consists of two families, voltage-dependent anion channel (VDAC) and Tom40, which are both located in the mitochondrial outer membrane. In Trypanosoma brucei, only a single member of the VDAC family has been described. We report the detection of two additional eukaryotic porin-like sequences in T. brucei. By bioinformatic means, we classify both as putative VDAC isoforms.