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Cell motility is universally driven by focal adhesion complexes (FAs) assembled in the cell envelope to connect the underlying substrate to cytoskeletal motors, generating traction forces in a highly regulated manner. In bacteria, focal adhesion complexes (bFAs) assembly is mediated by the Agl-Glt motility machinery, however how this machinery is connected to the cytoskeleton via cytoplasmic platform proteins, MglA and AglZ, remains unknown. Here, combining in vitro and single cell approaches, we show that the cytoplasmic region of the transmembrane GltJ protein contains two cytosolic motifs that independently recruit MglA-GTP (Linker) and AglZ (GYF), thus driving bFA assembly. Remarkably, binding of MglA-GTP causes a switch in the conformation of an adjacent Zinc finger domain (ZnR) that becomes available to recruit MglB, a MglA GTPase-Activating Protein. This binding activates GTP hydrolysis, which dissociates MglA from GltJ and is important to regulate bFA stability in vivo. These findings thus unravel the molecular mechanism of bFA assembly and regulation. Remarkably, the large number of other bacterial receptor proteins containing adjacent GYF and ZnR domains suggests conservation of this novel type of molecular switches.

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Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA., eLife digest Living organisms need energy to stay alive; in cells, this energy is supplied in the form of a small molecule called adenosine triphosphate, or ATP, a nucleotide that stores energy in the bonds between its three phosphate groups. ATP is present in all living cells and is often referred to as the energy currency of the cell, because it can be easily stored and transported to where it is needed. However, it is unknown why cells rely so heavily on ATP when a highly similar nucleotide called guanosine triphosphate, or GTP, could also act as an energy currency. There are several examples of proteins that originally used GTP and have since evolved to use ATP, but it is not clear why this switch occurred. One suggestion is that ATP is the more readily available nucleotide in the cell. To test this hypothesis, Updegrove, Harke et al. studied a protein that helps bacteria transition into spores, which are hardier and can survive in extreme environments until conditions become favorable for bacteria to grow again. In modern bacteria, this protein uses ATP to provide energy, but it evolved from an ancestral protein that used GTP instead. First, Updegrove, Harke et al. engineered the protein so that it became more similar to the ancestral protein and used GTP instead of ATP. When this was done, the protein gained the ability to break down GTP and release energy from it, but it no longer performed its enzymatic function. This suggests that both the energy released and the source of that energy are important for a protein’s activity. Further analysis showed that the modern version of the protein has evolved to briefly hold on to ATP after releasing its energy, which did not happen with GTP in the modified protein. Updegrove, Harke et al. also discovered that the levels of GTP in a bacterial cell fall as it transforms into a spore, while ATP levels remain relatively high. This suggests that ATP may indeed have become the source of energy of choice because it was more available. These findings provide insights into how ATP became the energy currency in cells, and suggest that how ATP is bound by proteins can impact a protein’s activity. Additionally, these experiments could help inform the development of drugs targeting proteins that bind nucleotides: it may be essential to consider the entirety of the binding event, and not just the release of energy.

Jumbo phages have attracted much attention by virtue of their extraordinary genome size and unusual aspects of biology. By performing a comparative genomics analysis of 224 jumbo phages, we suggest an objective inclusion criterion based on genome size distributions and present a synthetic overview of their manifold adaptations across major biological systems. By means of clustering and principal component analysis of the phyletic patterns of conserved genes, all known jumbo phages can be classified into three higher-order groups, which include both myoviral and siphoviral morphologies indicating multiple independent origins from smaller predecessors. Our study uncovers several under-appreciated or unreported aspects of the DNA replication, recombination, transcription and virion maturation systems. Leveraging sensitive sequence analysis methods, we identify novel protein-modifying enzymes that might help hijack the host-machinery. Focusing on host&ndash, virus conflicts, we detect strategies used to counter different wings of the bacterial immune system, such as cyclic nucleotide- and NAD+-dependent effector-activation, and prevention of superinfection during pseudolysogeny. We reconstruct the RNA-repair systems of jumbo phages that counter the consequences of RNA-targeting host effectors. These findings also suggest that several jumbo phage proteins provide a snapshot of the systems found in ancient replicons preceding the last universal ancestor of cellular life.

The phage shock protein (PSP) systems orchestrate a conserved stress response function by stabilizing the cell membrane and protecting bacteria from envelope stress. The full repertoire of PSP components remains poorly characterized. We combined comparative genomics and protein sequence-structure-function analyses to systematically identify homologs, phyletic patterns, domain architectures, and gene neighborhoods to trace the evolution of PSP components across the tree of life. This approach showed that the core component PspA/Snf7 (Psp/ESCRT systems) was present in the Last Universal Common Ancestor and that different clades co-opted a diverse range of partners to constitute distinct PSP systems. We identified several novel partners of the PSP system: (i) the Toastrack domain, which likely facilitates assembling diverse sub-membrane stress-sensing and signaling complexes, (ii) the newly-defined HAAS–PadR-like transcription regulator pair system, and (iii) multiple independent associations with ATPase or CesT/Tir-like chaperones, and Band-7 domain proteins that likely mediate sub-membrane dynamics. Our work also uncovered links between the PSP components and diverse SHOCT-like domains, suggesting a role in assembling membrane-associated complexes of proteins with disparate biochemical functions. Tracing the evolution of Psp cognate proteins provides new insights into the functions of the system and helps predict previously uncharacterized, often lineage-specific, membrane-dynamics and stress-response systems. The conservation of PSP systems across bacterial phyla emphasizes the importance of this stress response system in prokaryotes, while its modular diversity in various lineages indicates the emergence of lineage-specific cell-envelope structures, lifestyles, and adaptation mechanisms. The results can be accessed at https://jravilab.shinyapps.io/psp-evolution.

Manganese (Mn) is an essential trace nutrient for organisms because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016, and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and inductively coupled plasma-mass spectrometry (ICP-MS) to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negatively charged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch and found that the UPF0016 family of proteins has manganese export activity. We provide here the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, but rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand the available knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mn-responsive riboswitch can be used to identify new components of the manganese homeostasis machinery.

The phage shock protein (Psp) stress-response system protects bacteria from envelope stress through a cascade of interactions with other proteins and membrane lipids to stabilize the cell membrane. A key component of this multi-gene system is PspA, an effector protein that is found in diverse bacterial phyla, archaea, cyanobacteria, and chloroplasts. Other members of the Psp system include the cognate partners of PspA that are part of known operons: pspF||pspABC in Proteobacteria, liaIHGFSR in Firmicutes, and clgRpspAMN in Actinobacteria. Despite the functional significance of the Psp system, the conservation of PspA and other Psp functions, as well as the various genomic contexts of PspA, remain poorly characterized in Actinobacteria. Here we utilize a computational evolutionary approach to systematically identify the variations of the Psp system in ~450 completed actinobacterial genomes. We first determined the homologs of PspA and its cognate partners (as reported in Escherichia coli, Bacillus subtilis, and Mycobacterium tuberculosis) across Actinobacteria. This survey revealed that PspA and most of its functional partners are prevalent in Actinobacteria. We then found that PspA occurs in four predominant genomic contexts within Actinobacteria, the primary context being the clgRpspAM system previously identified in Mycobacteria. We also constructed a phylogenetic tree of PspA homologs (including paralogs) to trace the conservation and evolution of PspA across Actinobacteria. The genomic context revealed that PspA shows changes in its gene-neighborhood. The presence of multiple PspA contexts or of other known Psp members in genomic neighborhoods that do not carry pspA suggests yet undiscovered functional implications in envelope stress response mechanisms.

Cell motility is a central function of living cells, as it empowers colonization of new environmental niches, cooperation, and development of multicellular organisms. This process is achieved by complex yet precise energy-consuming machineries in both eukaryotes and bacteria. Bacteria move on surfaces using extracellular appendages such as flagella and pili but also by a less-understood process called gliding motility. During this process, rod-shaped bacteria move smoothly along their long axis without any visible morphological changes besides occasional bending. For this reason, the molecular mechanism of gliding motility and its origin have long remained a complete mystery. An important breakthrough in the understanding of gliding motility came from single cell and genetic studies in the delta-proteobacterium Myxococcus xanthus. These early studies revealed, for the first time, the existence of bacterial Focal Adhesion complexes (FA). FAs are formed at the bacterial pole and rapidly move towards the opposite cell pole. Their attachment to the underlying surface is linked to cell propulsion, in a process similar to the rearward translocation of actomyosin complexes in Apicomplexans. The protein machinery that forms at FAs was shown to contain up to seventeen proteins predicted to localize in all layers of the bacterial cell envelope, the cytosolic face, the inner membrane (IM), the periplasmic space and the outer membrane (OM). Among these proteins, a proton-gated channel at the inner membrane was identified as the molecular motor. Thus, thrust generation requires the transduction of traction forces generated at the inner membrane through the cell envelope beyond the rigid barrier of the bacterial peptidoglycan.

The intraerythrocytic apicomplexan Babesia microti , the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-length B. microti AMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including other Babesia and Theileria species, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed in Escherichia coli reacted with a mouse anti-BmAMA1 antibody using Western blotting. In vitro binding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation of B. microti parasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

Spores of Bacillus subtilis are dormant cell types that are formed when the bacterium encounters starvation conditions. Spores are encased in a shell, termed the coat, which is composed of approximately seventy different proteins and protects the spore’s genetic material from environmental insults. The structural component of the basement layer of the coat is an exceptional cytoskeletal protein, termed SpoIVA, which binds and hydrolyzes ATP. ATP hydrolysis is utilized to drive a conformational change in SpoIVA that leads to its irreversible self-assembly into a static polymer in vitro. Here, we characterize the middle domain of SpoIVA, the predicted secondary structure of which resembles the chemotaxis protein CheX but, unlike CheX, does not harbor residues required for phosphatase activity. Disruptions in this domain did not abolish ATP hydrolysis, but resulted in mislocalization of the protein and reduction in sporulation efficiency in vivo. In vitro, disruptions in this domain prevented the ATP hydrolysis-driven conformational change in SpoIVA required for polymerization and led to the aggregation of SpoIVA into particles that did not form filaments. We propose a model in which SpoIVA initially assumes a conformation in which it inhibits its own aggregation into particles, and that ATP hydrolysis remodels the protein so that it assumes a polymerization-competent conformation.

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

Exposure to ionizing radiation has a profound effect on the viability and further replication of living cells. Radiation is used for the treatment of cancer [1], generation of attenuated vaccines [2], and in food safety [3]. Radiation attenuation has also been one of the earliest methods used to generate whole parasite–based malaria vaccines [4–6]. Given the importance of ionizing radiation in the treatment of different forms of cancer, a large body of scientific data has accumulated on the effects of ionizing radiation on the cell cycle [7]. Such studies have shed light on the molecular and biochemical aspects of radiation-mediated cell death and survival mechanisms [1, 8]. These studies have also provided insight into the cell cycle checkpoints and the complex signaling pathways and associated molecules that ensure that each phase of the cell cycle is completed without errors [7, 9, 10]. Ionizing radiation is detrimental to the growth and survival of Plasmodium parasites. In murine models, asexual blood-stage parasites exposed to high doses of radiation failed to cause blood-stage parasite infection [11], and immunization with radiation-attenuated asexual-stage parasites protected against parasitemia [11–13] and experimental cerebral malaria [11]. In preerythrocytic-stage malaria, exposure of Plasmodium sporozoites to a threshold level of γ-radiation allows their invasion into liver cells and early development. However, these parasites undergo arrested development and fail to produce infectious liver-stage merozoites and, consequently, blood-stage parasite infection [14]. Irradiation-attenuated malaria sporozoites protect mice [4, 15] and humans [5, 16] against malaria in experimental challenge studies. Accordingly, despite the limited success of subunit-based malaria vaccines in clinical testing [17], there has been considerable interest in using radiation exposure to create an attenuated sporozoite-stage vaccine [14, 16]. In this study, we measured the effects of different doses of γ-irradiation on P. falciparum in short-term and long-term cultures. These studies were performed to evaluate both the immediate effect on survival and the ability of rare parasite populations to recover and repopulate. We also studied the effect of γ-irradiation on parasite structure, by fluorescence microscopy and transmission electron microscopy. In addition, we performed extensive genome-wide transcriptional profiling in P. falciparum exposed to increasing doses of γ-radiation, to identify parasite proteins that could be correlated with survival and cell death, and, on the basis of the data set, created a functionally diverse molecular signature of growth attenuation. Last, bioinformatic analyses were performed to decipher the biological systems altered by γ-irradiation that are associated with growth regulation and survival.

Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood captures the molecular and immunological events associated with the pathogenesis of disease. We find that during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore, our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory markers. Complement component C1q, β-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell antigen, DnaJC, member 15, glutathione S -transferase omega-1, and thymidine kinase 1 were overexpressed in blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM in blood. A subset of these mediators of ECM warrant validation in P. falciparum -infected young African children as diagnostic markers of CM.

Cerebral malaria (CM) is a primary cause of malaria-associated deaths among young African children. Yet no diagnostic tools are available that could be used to predict which of the children infected withPlasmodium falciparummalaria will progress to CM. We used thePlasmodium bergheiANKA murine model of experimental cerebral malaria (ECM) and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of ECM. Comparative expression analyses were performed with C57BL/6 mice, which have an ECM-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin α1 proteins by Western blot analysis than mice unaffected by ECM, suggesting the possible utility of these molecules as prognostic biomarkers of CM in humans. We suggest that the higher expression of hemoglobin α1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM.

We describe AMIN (Amidase N-terminal domain), a novel protein domain found specifically in bacterial periplasmic proteins. AMIN domains are widely distributed among peptidoglycan hydrolases and transporter protein families. Based on experimental data, contextual information and phyletic profiles, we suggest that AMIN domains mediate the targeting of periplasmic or extracellular proteins to specific regions of the bacterial envelope. Contact: fgueiros@iq.usp.br Supplementary information: Supplementary data are available at Bioinformatics online.

Comparative genomics of parasitic protists and their free-living relatives are profoundly impacting our understanding of the regulatory systems involved in transcription and chromatin dynamics. While some parts of these systems are highly conserved, other parts are rapidly evolving, thereby providing the molecular basis for the variety in the regulatory adaptations of eukaryotes. The gross number of specific transcription factors and chromatin proteins are positively correlated with proteome size in eukaryotes. However, the individual types of specific transcription factors show an enormous variety across different eukaryotic lineages. The dominant families of specific transcription factors even differ between sister lineages, and have been shaped by gene loss and lineage-specific expansions. Recognition of this principle has helped in identifying the hitherto unknown, major specific transcription factors of several parasites, such as apicomplexans, Entamoeba histolytica, Trichomonas vaginalis, Phytophthora and ciliates. Comparative analysis of predicted chromatin proteins from protists allows reconstruction of the early evolutionary history of histone and DNA modification, nucleosome assembly and chromatin-remodeling systems. Many key catalytic, peptide-binding and DNA-binding domains in these systems ultimately had bacterial precursors, but were put together into distinctive regulatory complexes that are unique to the eukaryotes. In the case of histone methylases, histone demethylases and SWI2/SNF2 ATPases, proliferation of paralogous families followed by acquisition of novel domain architectures, seem to have played a major role in producing a diverse set of enzymes that create and respond to an epigenetic code of modified histones. The diversification of histone acetylases and DNA methylases appears to have proceeded via repeated emergence of new versions, most probably via transfers from bacteria to different eukaryotic lineages, again resulting in lineage-specific diversity in epigenetic signals. Even though the key histone modifications are universal to eukaryotes, domain architectures of proteins binding post-translationally modified-histones vary considerably across eukaryotes. This indicates that the histone code might be “interpreted” differently from model organisms in parasitic protists and their relatives. The complexity of domain architectures of chromatin proteins appears to have increased during eukaryotic evolution. Thus, Trichomonas, Giardia, Naegleria and kinetoplastids have relatively simple domain architectures, whereas apicomplexans and oomycetes have more complex architectures. RNA-dependent post-transcriptional silencing systems, which interact with chromatin-level regulatory systems, show considerable variability across parasitic protists, with complete loss in many apicomplexans and partial loss in Trichomonas vaginalis. This evolutionary synthesis offers a robust scaffold for future investigation of transcription and chromatin structure in parasitic protists.

We expand the functionally uncharacterized DOMON domain superfamily to identify several novel families, including the first prokaryotic representatives. Using several computational tools we show that it is involved in ligand binding—either as heme- or sugar-binding domains. We present evidence that the DOMON domain along with the DM13 domain comprises a novel electron-transfer system potentially involved in oxidative modification of animal cell-surface proteins. Other novel versions might function as sugar sensors of histidine kinases of bacterial two component systems.Contact: aravind@ncbi.nlm.nih.gov or aravind@mail.nih.govSupplementary information: Supplementary data are available at Bioinformatics online and also at ftp://ftp.ncbi.nih.gov/pub/aravind/domon/.

Using structural comparisons, we identified a novel domain with a simple fold in the bacterial cell division ATPase FtsA, the archaeo-eukaryotic RNA polymerase subunit Rpb7p, the GyrI superfamily, and the uncharacterized MTH1598/Tm1083-like proteins. The fold contains a core of 3 strands, forming a curved sheet, and a single helix in a strand-helix-strand-strand (SHS2) configuration. The SHS2 domain may exist either in single or duplicate copies within the same polypeptide. The single-copy versions of the domain in FtsA and Rbp7p are most closely related, and appear to mediate protein-protein interactions by means of strand 1, and the loop between strand 2 and strand 3 of the domain. We predict that the interactions between FtsA and its functional partners in bacterial cell division are likely to be similar to the interactions of Rbp7p in the archaeo-eukaryotic RNA polymerase complex. The dimeric versions typified by the GyrI superfamily appear to have been adapted for small-molecule binding. Sequence profiles searches helped us to identify several new versions of the GyrI superfamily, including a family of secreted forms that is found only in animals and the bacterial pathogen Leptospira. Through sequence-structure comparisons, we predict the positions that are likely to be important for ligand specificity in the GyrI superfamily. In the MTH1598/Tm1083-like proteins, a SHS2 domain is inserted into the loop between strand 1 and helix 1 of another SHS2 domain. This has resulted in a structure that has convergent similarities with the Hsp33 and green fluorescent protein folds. The sequence conservation pattern and its phyletic profile suggest that it might function as an enzyme in some conserved aspect of nucleic acid metabolism. Thus, the SHS2 domain is an example of a simple module that has been adapted to perform an entire spectrum of functions ranging from protein-protein interactions to small-molecule recognition and catalysis.

Protein sequence and structure comparisons show that the catalytic domains of Class I aminoacyl-tRNA synthetases, a related family of nucleotidyltransferases involved primarily in coenzyme biosynthesis, nucleotide-binding domains related to the UspA protein (USPA domains), photolyases, electron transport flavoproteins, and PP-loop-containing ATPases together comprise a distinct class of α/β domains designated the HUP domain after HIGH-signature proteins, UspA, and PP-ATPase. Several lines of evidence are presented to support the monophyly of the HUP domains, to the exclusion of other three-layered α/β folds with the generic “Rossmann-like” topology. Cladistic analysis, with patterns of structural and sequence similarity used as discrete characters, identified three major evolutionary lineages within the HUP domain class: the PP-ATPases; the HIGH superfamily, which includes class I aaRS and related nucleotidyltransferases containing the HIGH signature in their nucleotide-binding loop; and a previously unrecognized USPA-like group, which includes USPA domains, electron transport flavoproteins, and photolyases. Examination of the patterns of phyletic distribution of distinct families within these three major lineages suggests that the Last Universal Common Ancestor of all modern life forms encoded 15–18 distinct α/β ATPases and nucleotide-binding proteins of the HUP class. This points to an extensive radiation of HUP domains before the last universal common ancestor (LUCA), during which the multiple class I aminoacyl-tRNA synthetases emerged only at a late stage. Thus, substantial evolutionary diversification of protein domains occurred well before the modern version of the protein-dependent translation machinery was established, i.e., still in the RNA world. Proteins 2002;48:1–14. © 2002 Wiley-Liss, Inc.

Using computational analysis, a novel superfamily of beta-strand-rich domains was identified in the Molybdenum cofactor sulfurase and several other proteins from both prokaryotes and eukaryotes. These MOSC domains contain an absolutely conserved cysteine and occur either as stand-alone forms such as the bacterial YiiM proteins, or fused to other domains such as a NifS-like catalytic domain in Molybdenum cofactor sulfurase. The MOSC domain is predicted to be a sulfur-carrier domain that receives sulfur abstracted by the pyridoxal phosphate-dependent NifS-like enzymes, on its conserved cysteine, and delivers it for the formation of diverse sulfur-metal clusters. The identification of this domain may clarify the mechanism of biogenesis of various metallo-enzymes including Molybdenum cofactor-containing enzymes that are compromised in human type II xanthinuria.

The highly structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitches for which the cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis, and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests that the genes that are preceded by this motif and encode a diverse set of poorly characterized membrane proteins have roles in metal homeostasis.

CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold). Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and “effector” domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.

A novel, 200–230 amino acid extracellular domain was identified in the plant cytokinin receptor Cre1, in the receptor-histidine kinase DhkA and the adenylyl cyclase Acg from the slime mold Dictyostelium discoideum, and in a variety of other receptor-like proteins from bacteria and eukaryotes. The domain is predicted to bind diverse low molecular weight ligands, such as the cytokinin-like adenine derivatives or peptides, and mediate signal transduction through the respective receptors.

Central cellular functions such as metabolism, solute transport and signal transduction are regulated, in part, via binding of small molecules by specialized domains. Using sensitive methods for sequence profile analysis and protein structure comparison, we exhaustively surveyed the protein sets from completely sequenced genomes for all occurrences of 21 intracellular small-molecule-binding domains (SMBDs) that are represented in at least two of the three major divisions of life (bacteria, archaea and eukaryotes). These included previously characterized domains such as PAS, GAF, ACT and ferredoxins, as well as three newly predicted SMBDs, namely the 4-vinyl reductase (4VR) domain, the NIFX domain and the 3-histidines (3H) domain. Although there are only a limited number of different superfamilies of these ancient SMBDs, they are present in numerous distinct proteins combined with various enzymatic, transport and signal-transducing domains. Most of the SMBDs show considerable evolutionary mobility and are involved in the generation of many lineage-specific domain architectures. Frequent re-invention of analogous architectures involving functionally related, but not homologous, domains was detected, such as, fusion of different SMBDs to several types of DNA-binding domains to form diverse transcription regulators in prokaryotes and eukaryotes. This is suggestive of similar selective forces affecting the diverse SMBDs and resulting in the formation of multidomain proteins that fit a limited number of functional stereotypes. Using the "guilt by association approach", the identification of SMBDs allowed prediction of functions and mode of regulation for a variety of previously uncharacterized proteins.

A bacterial assay was developed for testing HTLV-1 reverse transcriptase sensitivity to common nucleoside analog inhibitors in an Escherichia coli strain characterized by a temperature sensitive PolI/RecA deletion phenotype. This genetic complementation assay exploits the ability of HTLV-1 reverse transcriptase to functionally replace these missing activities at nonpermissive temperatures. The four inhibitors tested, dideoxyinosine, dideoxyadenosine, deoxythymidine, and didehydrodeoxythymidine are well-known inhibitors of HIV reverse transcriptase. All except dideoxyadenosine showed a strong activity against HTLV-1 reverse transcriptase with IC(50); in the nanomolar range. Sequence alignments were used to identify amino acid residues in HTLV-1 reverse transcriptase, which correspond to those identified as important for drug-resistance in HIV reverse transcriptase. Mutations of some of these HTLV-1 residues altered the IC(50) for the inhibitors as expected, which suggests that these amino acids have a function in HTLV-1 reverse transcriptase similar to that of their homologs in HIV reverse transcriptase. Copyright 2000 Academic Press.

Background The virus-host arms race is a major theater for evolutionary innovation. Archaea and bacteria have evolved diverse, elaborate antivirus defense systems that function on two general principles: i) immune systems that discriminate self DNA from nonself DNA and specifically destroy the foreign, in particular viral, genomes, whereas the host genome is protected, or ii) programmed cell suicide or dormancy induced by infection. Presentation of the hypothesis Almost all genomic loci encoding immunity systems such as CRISPR-Cas, restriction-modification and DNA phosphorothioation also encompass suicide genes, in particular those encoding known and predicted toxin nucleases, which do not appear to be directly involved in immunity. In contrast, the immunity systems do not appear to encode antitoxins found in typical toxin-antitoxin systems. This raises the possibility that components of the immunity system themselves act as reversible inhibitors of the associated toxin proteins or domains as has been demonstrated for the Escherichia coli anticodon nuclease PrrC that interacts with the PrrI restriction-modification system. We hypothesize that coupling of diverse immunity and suicide/dormancy systems in prokaryotes evolved under selective pressure to provide robustness to the antivirus response. We further propose that the involvement of suicide/dormancy systems in the coupled antivirus response could take two distinct forms: 1) induction of a dormancy-like state in the infected cell to ‘buy time’ for activation of adaptive immunity; 2) suicide or dormancy as the final recourse to prevent viral spread triggered by the failure of immunity. Testing the hypothesis This hypothesis entails many experimentally testable predictions. Specifically, we predict that Cas2 protein present in all cas operons is a mRNA-cleaving nuclease (interferase) that might be activated at an early stage of virus infection to enable incorporation of virus-specific spacers into the CRISPR locus or to trigger cell suicide when the immune function of CRISPR-Cas systems fails. Similarly, toxin-like activity is predicted for components of numerous other defense loci. Implications of the hypothesis The hypothesis implies that antivirus response in prokaryotes involves key decision-making steps at which the cell chooses the path to follow by sensing the course of virus infection. Reviewers This article was reviewed by Arcady Mushegian, Etienne Joly and Nick Grishin. For complete reviews, go to the Reviewers’ reports section.

The mode of action of the bacterial ter cluster and TelA genes, implicated in natural resistance to tellurite and other xenobiotic toxic compounds, pore-forming colicins and several bacteriophages, has remained enigmatic for almost two decades. Using comparative genomics, sequence-profile searches and structural analysis we present evidence that the ter gene products and their functional partners constitute previously underappreciated, chemical stress response and anti-viral defense systems of bacteria. Based on contextual information from conserved gene neighborhoods and domain architectures, we show that the ter gene products and TelA lie at the center of membrane-linked metal recognition complexes with regulatory ramifications encompassing phosphorylation-dependent signal transduction, RNA-dependent regulation, biosynthesis of nucleoside-like metabolites and DNA processing. Our analysis suggests that the multiple metal-binding and non-binding TerD paralogs and TerC are likely to constitute a membrane-associated complex, which might also include TerB and TerY, and feature several, distinct metal-binding sites. Versions of the TerB domain might also bind small molecule ligands and link the TerD paralog-TerC complex to biosynthetic modules comprising phosphoribosyltransferases (PRTases), ATP grasp amidoligases, TIM-barrel carbon-carbon lyases, and HAD phosphoesterases, which are predicted to synthesize novel nucleoside-like molecules. One of the PRTases is also likely to interact with RNA by means of its Pelota/Ribosomal protein L7AE-like domain. The von Willebrand factor A domain protein, TerY, is predicted to be part of a distinct phosphorylation switch, coupling a protein kinase and a PP2C phosphatase. We show, based on the evidence from numerous conserved gene neighborhoods and domain architectures, that both the TerB and TelA domains have been linked to diverse lipid-interaction domains, such as two novel PH-like and the Coq4 domains, in different bacteria, and are likely to comprise membrane-associated sensory complexes that might additionally contain periplasmic binding-protein-II and OmpA domains. We also show that the TerD and TerB domains and the TerY-associated phosphorylation system are functionally linked to many distinct DNA-processing complexes, which feature proteins with SWI2/SNF2 and RecQ-like helicases, multiple AAA+ ATPases, McrC-N-terminal domain proteins, several restriction endonuclease fold DNases, DNA-binding domains and a type-VII/Esx-like system, which is at the center of a predicted DNA transfer apparatus. These DNA-processing modules and associated genes are predicted to be involved in restriction or suicidal action in response to phages and possibly repairing xenobiotic-induced DNA damage. In some eukaryotes, certain components of the ter system appear to be recruited to function in conjunction with the ubiquitin system and calcium-signaling pathways.

B cells and plasma cells possess distinct RNA processing environments that respectively promote the expression of membrane-associated Ig by B cells versus the secretion of Ig by plasma cells. Through a combination of transcriptional profiling and screening using a lentiviral short-hairpin RNA interference library, we show that both the splicing factor hnRNPLL and the transcription elongation factor ELL2 modulate the ratio of secreted versus membrane-encoding Ighg2b transcripts in MPC11 plasmacytoma cell lines. hnRNPLL and ELL2 are both highly expressed in primary plasma cells relative to B cells, but hnRNPLL binds Ighg2b mRNA transcripts and promotes an increase in levels of the membrane-encoding Ighg2b isoform at the expense of the secreted Ighg2b isoform, whereas ELL2 counteracts this effect and drives Ig secretion by increasing the frequency of the secreted Ighg2b isoform. As in T cells, hnRNPLL also alters the splicing pattern of mRNA encoding the adhesion receptor CD44, promoting exon inclusion, and decreasing the overall level of CD44 expression. Further characterization of ELL2-dependent transcription by RNA-Seq revealed that ∼12% of transcripts expressed by plasma cells were differentially processed because of the activities of ELL2, including B-cell maturation antigen BCMA, a receptor with a defined role in plasma cell survival. Taken together, our data identify hnRNPLL and ELL2 as regulators of pre-mRNA processing in plasma cells.

Background Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized “Photorhabdus virulence cassettes (PVC)”, PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative ‘cheating’ in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers. Reviewers This article was reviewed by AM, FE and IZ.

Background Almost all known nucleic acid polymerases catalyze 5'-3' polymerization by mediating the attack on an incoming nucleotide 5' triphosphate by the 3'OH from the growing polynucleotide chain in a template dependent or independent manner. The only known exception to this rule is the Thg1 RNA polymerase that catalyzes 3'-5' polymerization in vitro and also in vivo as a part of the maturation process of histidinyl tRNA. While the initial reaction catalyzed by Thg1 has been compared to adenylation catalyzed by the aminoacyl tRNA synthetases, the evolutionary relationships of Thg1 and the actual nature of the polymerase reaction catalyzed by it remain unclear. Results Using sensitive profile-profile comparison and structure prediction methods we show that the catalytic domain Thg1 contains a RRM (ferredoxin) fold palm domain, just like the viral RNA-dependent RNA polymerases, reverse transcriptases, family A and B DNA polymerases, adenylyl cyclases, diguanylate cyclases (GGDEF domain) and the predicted polymerase of the CRISPR system. We show just as in these polymerases, Thg1 possesses an active site with three acidic residues that chelate Mg++ cations. Based on this we predict that Thg1 catalyzes polymerization similarly to the 5'-3' polymerases, but uses the incoming 3' OH to attack the 5' triphosphate generated at the end of the elongating polynucleotide. In addition we identify a distinct set of residues unique to Thg1 that we predict as comprising a second active site, which catalyzes the initial adenylation reaction to prime 3'-5' polymerization. Based on contextual information from conserved gene neighborhoods we show that Thg1 might function in conjunction with a polynucleotide kinase that generates an initial 5' phosphate substrate for it at the end of a RNA molecule. In addition to histidinyl tRNA maturation, Thg1 might have other RNA repair roles in representatives from all the three superkingdoms of life as well as certain large DNA viruses. We also present evidence that among the polymerase-like domains Thg1 is most closely related to the catalytic domains of the GGDEF and CRISPR polymerase proteins. Conclusion Based on this relationship and the phyletic patterns of these enzymes we infer that the Thg1 protein is likely to represent an archaeo-eukaryotic branch of the same clade of proteins that gave rise to the mobile CRISPR polymerases and in bacteria spawned the GGDEF domains. Thg1 is likely to be close to the ancestral version of this family of enzymes that might have played a role in RNA repair in the last universal common ancestor. Reviewers This article was reviewed by S. Balaji and V.V. Dolja.

An in-depth knowledge of the host molecules and biological pathways that contribute towards the pathogenesis of cerebral malaria would help guide the development of novel prognostics and therapeutics. Genome-wide transcriptional profiling of the brain tissue during experimental cerebral malaria (ECM ) caused by Plasmodium berghei ANKA parasites in mice, a well established surrogate of human cerebral malaria, has been useful in predicting the functional classes of genes involved and pathways altered during the course of disease. To further understand the contribution of individual genes to the pathogenesis of ECM, we examined the biological relevance of three molecules -- CD14, galectin-3, and OX40 that were previously shown to be overexpressed during ECM. We find that CD14 plays a predominant role in the induction of ECM and regulation of parasite density; deletion of the CD14 gene not only prevented the onset of disease in a majority of susceptible mice (only 21% of CD14-deficient compared to 80% of wildtype mice developed ECM, p

The genomic revolution has provided the first glimpses of the architecture of regulatory networks. Combined with evolutionary information, the “network view” of life processes leads to remarkable insights into how biological systems have been shaped by various forces. This understanding is critical because biological systems, including regulatory networks, are not products of engineering but of historical contingencies. In this light, we attempt a synthetic overview of the natural history of regulatory networks operating in the development and differentiation of multicellular organisms. We first introduce regulatory networks and their organizational principles as can be deduced using ideas from the graph theory. We then discuss findings from comparative genomics to illustrate the effects of lineage-specific expansions, gene-loss, and non-protein-coding DNA on the architecture of networks. We consider the interaction between expansions of transcription factors, and cis regulatory and more general chromatin state stabilizing elements in the emergence of morphological complexity. Finally, we consider a case study of the Notch sub-network, which is present throughout Metazoa, to examine how such a regulatory system has been pieced together in evolution from new innovations and pre-existing components that were originally functionally distinct.

Deleted in Breast Cancer-1 (DBC1) and its paralog CARP-1 are large multi-domain proteins, with a nuclear or perinuclear localization, and a role in promoting apoptosis upon processing by caspases. Recent studies on human DBC1 show that it is a specific inhibitor of the sirtuin-type deacetylase, Sirt1, which deacetylates histones and p53. Using sensitive sequence profile searches and HMM-HMM comparisons we show that the central conserved globular domain present in the DBC1 and it homologs from diverse eukaryotes is a catalytically inactive version of the Nudix hydrolase (MutT) domain. Given that Nudix domains are known to bind nucleoside diphosphate sugars and NAD, we predict that this domain in DBC1 and its homologs binds NAD metabolites such as ADP-ribose. Hence, we propose that DBC1 and its homologs are likely to regulate the activity of SIRT1 or related deacetylases by sensing the soluble products or substrates of the NAD-dependent deacetylation reaction. The complex domain architectures of the members of the DBC1 family, which include fusions to the RNA-binding S1-like domain, the DNA-binding SAP domain and EF-hand domains, suggest that they are likely to function as integrators of distinct regulatory signals including chromatin protein modification, soluble compounds in NAD metabolism, apoptotic stimuli and RNA recognition.

Apicomplexa have developed distinctive adaptations for invading and surviving within animal cells. Here a synthetic overview of the diversity and evolutionary history of cell membrane-associated, -secreted, and -exported proteins related to apicomplexan parasitism is presented. A notable feature in this regard was the early acquisition of adhesion protein domains and glycosylation systems through lateral transfer from animals. These were utilized in multiple contexts, including invasion of host cells and parasite-specific developmental processes. Apicomplexans possess a specialized version of the ancestral alveolate extrusion machinery, the rhoptries and micronemes, which are deployed in invasion and delivery of proteins into host cells. Each apicomplexan lineage has evolved a unique spectrum of extruded proteins that modify host molecules in diverse ways. Hematozoans, in particular, appear to have evolved novel systems for export of proteins into the host organelles and cell membrane during intracellular development. These exported proteins are an important aspect of the pathogenesis of Plasmodium and Theileria, being involved in response to fever and in leukocyte proliferation respectively. The complement of apicomplexan surface proteins has primarily diversified via massive lineage-specific expansions of certain protein families, which are often coded by subtelomeric gene arrays. Many of these families have been found to be central to immune evasion. Domain shuffling and accretion have resulted in adhesins with new domain architectures. In terms of individual genes, constant selective pressures from the host immune response has resulted in extensive protein polymorphisms and gene losses. Apicomplexans have also evolved complex regulatory mechanisms controlling expression and maturation of surface proteins at the chromatin, transcriptional, posttranscriptional, and posttranslational levels. Evolutionary reconstruction suggests that the ancestral apicomplexan had thrombospondin and EGF domain adhesins, which were linked to the parasite cytoskeleton, and played a central role in invasion through formation of the moving junction. It also suggests that the ancestral parasite had O-linked glycosylation of surface proteins which was partially or entirely lost in hematozoan lineages.

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as “hypothetical.” We find that cultivation of P. falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the “crisis forms” and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.

Apicomplexa have developed distinctive adaptations for invading and surviving within animal cells. Here a synthetic overview of the diversity and evolutionary history of cell membrane‐associated, ‐secreted, and ‐exported proteins related to apicomplexan parasitism is presented. A notable feature in this regard was the early acquisition of adhesion protein domains and glycosylation systems through lateral transfer from animals. These were utilized in multiple contexts, including invasion of host cells and parasite‐specific developmental processes. Apicomplexans possess a specialized version of the ancestral alveolate extrusion machinery, the rhoptries and micronemes, which are deployed in invasion and delivery of proteins into host cells. Each apicomplexan lineage has evolved a unique spectrum of extruded proteins that modify host molecules in diverse ways. Hematozoans, in particular, appear to have evolved novel systems for export of proteins into the host organelles and cell membrane during intracellular development. These exported proteins are an important aspect of the pathogenesis of Plasmodium and Theileria , being involved in response to fever and in leukocyte proliferation respectively. The complement of apicomplexan surface proteins has primarily diversified via massive lineage‐specific expansions of certain protein families, which are often coded by subtelomeric gene arrays. Many of these families have been found to be central to immune evasion. Domain shuffling and accretion have resulted in adhesins with new domain architectures. In terms of individual genes, constant selective pressures from the host immune response has resulted in extensive protein polymorphisms and gene losses. Apicomplexans have also evolved complex regulatory mechanisms controlling expression and maturation of surface proteins at the chromatin, transcriptional, posttranscriptional, and posttranslational levels. Evolutionary reconstruction suggests that the ancestral apicomplexan had thrombospondin and EGF domain adhesins, which were linked to the parasite cytoskeleton, and played a central role in invasion through formation of the moving junction. It also suggests that the ancestral parasite had O‐linked glycosylation of surface proteins which was partially or entirely lost in hematozoan lineages.

Background The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. Results Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S)-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix. Conclusion Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream signaling domains. However, upon occurrence of specific conformational changes due to binding of ligand or other sensory inputs in a linked upstream domain it transmits the signal to the downstream domain. Thus, the S-helix represents one of the most prevalent functional themes involved in the flow of signals between modules in diverse prokaryote-type multi-domain signaling proteins. Reviewers This article was reviewed by Frank Eisenhaber, Arcady Mushegian and Sandor Pongor.