Structural analysis of Brucella abortus RicA substitutions that do not impair interaction with human Rab2 GTPase.

BACKGROUND: Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen. RESULTS: In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the "absence of interference" approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a "negative image" of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the "beta helix" or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that "absence of interference" approach also generates surfaces that could be necessary for folding. CONCLUSION: Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed ß-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.

The centenary of Janssens's chiasmatype theory.

The segregation and random assortment of characters observed by Mendel have their basis in the behavior of chromosomes in meiosis. But showing this actually to be the case requires a correct understanding of the meiotic behavior of chromosomes. This was achieved only gradually, over several decades, with much dispute and confusion along the way. One crucial step in the understanding of meiosis was provided in 1909 by Frans Alfons Janssens who published in La Cellule an article entitled "La théorie de la Chiasmatypie. Nouvelle interprétation des cinèses de maturation," which contains the first description of the chiasma structure. He observed that, of the four chromatids present at the connection sites (chiasmata sites) at diplotene or anaphase of the first meiotic division, two crossed each other and two did not. He therefore postulated that the maternal and paternal chromatids that crossed penetrated the other until they broke and rejoined in maternal and paternal segments new ways; the other two chromatids remained free and thus intact. This allowed him also to propose that the chromatids distributed in the four nuclei issued from the second meiotic division had various combinations of maternal and paternal segments of each chromosome. And conversely, permitted the appreciation that the laws of Mendelian segregation required breakage and joining (crossing over) between homologous non-sister chromatids. Although Janssens's article found a broad appreciative audience and had a large influence on the chromosomal theory at that time, his theory was resisted by both geneticists and cytologists for several decades. This Perspectives aims to highlight the novelty of Janssens's chiasmatype theory by examining the historical background and our actual understanding of meiotic recombination.

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.

Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.

Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-ß-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a ΔvirB mutant. In HeLa cells, a ΔricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusΔricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.

A gene-specific requirement of RNA polymerase II CTD phosphorylation for sexual differentiation in S. pombe.

BACKGROUND: The switch from cellular proliferation to differentiation occurs to a large extent through specific programs of gene expression. In fission yeast, the master regulator of sexual differentiation, ste11, is induced by environmental conditions leading to mating and meiosis. RESULTS: We show that phosphorylation of serine 2 (S2P) in the C-terminal domain of the largest subunit of the RNA polymerase II (PolII) enzyme by the Lsk1 cyclin-dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. The recruitment of the Lsk1 kinase initiates in the vicinity of the transcription start site of ste11, resulting in a marked increase of S2P on the ste11 unit, including an extended 5' untranslated region (5'UTR). This pattern contrasts with the classical gradient of S2P toward the 3' region. In the absence of S2P, both PolII occupancy at the ste11 locus and ste11 expression are impaired. This results in sterility, which is rescued by expression of the ste11 coding sequence from the adh1 promoter. CONCLUSION: Thus, the S2P polymerase plays a specific, regulatory role in cell differentiation through the induction of ste11.

'Edgetic' perturbation of a C. elegans BCL2 ortholog.

Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

Xylanase XYL1p from Scytalidium acidophilum: site-directed mutagenesis and acidophilic adaptation.

The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH's and temperatures were determined. All mutations increased the pH optimum by 0.5-1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T(m) maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.

Empirically controlled mapping of the Caenorhabditis elegans protein-protein interactome network.

To provide accurate biological hypotheses and elucidate global properties of cellular networks, systematic identification of protein-protein interactions must meet high quality standards.We present an expanded C. elegans protein-protein interaction network, or 'interactome' map, derived from testing a matrix of approximately 10,000 x approximately 10,000 proteins using a highly specific, high-throughput yeast two-hybrid system. Through a new empirical quality control framework, we show that the resulting data set (Worm Interactome 2007, or WI-2007) was similar in quality to low-throughput data curated from the literature. We filtered previous interaction data sets and integrated them with WI-2007 to generate a high-confidence consolidated map (Worm Interactome version 8, or WI8). This work allowed us to estimate the size of the worm interactome at approximately 116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationships between genes or proteins

Literature-curated protein interaction datasets.

High-quality datasets are needed to understand how global and local properties of protein-protein interaction, or 'interactome', networks relate to biological mechanisms, and to guide research on individual proteins. In an evaluation of existing curation of protein interaction experiments reported in the literature, we found that curation can be error-prone and possibly of lower quality than commonly assumed.

An experimentally derived confidence score for binary protein-protein interactions.

Information on protein-protein interactions is of central importance for many areas of biomedical research. At present no method exists to systematically and experimentally assess the quality of individual interactions reported in interaction mapping experiments. To provide a standardized confidence-scoring method that can be applied to tens of thousands of protein interactions, we have developed an interaction tool kit consisting of four complementary, high-throughput protein interaction assays. We benchmarked these assays against positive and random reference sets consisting of well documented pairs of interacting human proteins and randomly chosen protein pairs, respectively. A logistic regression model was trained using the data from these reference sets to combine the assay outputs and calculate the probability that any newly identified interaction pair is a true biophysical interaction once it has been tested in the tool kit. This general approach will allow a systematic and empirical assignment of confidence scores to all individual protein-protein interactions in interactome networks.

The conserved Wobble uridine tRNA thiolase Ctu1-Ctu2 is required to maintain genome integrity.

Modified nucleosides close to the anticodon are important for the proper decoding of mRNA by the ribosome. Particularly, the uridine at the first anticodon position (U34) of glutamate, lysine, and glutamine tRNAs is universally thiolated (S(2)U34), which is proposed to be crucial for both restriction of wobble in the corresponding split codon box and efficient codon-anticodon interaction. Here we show that the highly conserved complex Ctu1-Ctu2 (cytosolic thiouridylase) is responsible for the 2-thiolation of cytosolic tRNAs in the nematode and fission yeast. In both species, inactivation of the complex leads to loss of thiolation on tRNAs and to a thermosensitive decrease of viability associated with marked ploidy abnormalities and aberrant development. Increased level of the corresponding tRNAs suppresses the fission yeast defects, and our data suggest that these defects could result from both misreading and frame shifting during translation. Thus, a translation defect due to unmodified tRNAs results in severe genome instability.

Recruitment of P-TEFb (Cdk9-Pch1) to chromatin by the cap-methyl transferase Pcm1 in fission yeast.

Capping of nascent pre-mRNAs is thought to be a prerequisite for productive elongation and associated serine 2 phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (PolII). The mechanism mediating this link is unknown, but is likely to include the capping machinery and P-TEPb. We report that the fission yeast P-TEFb (Cdk9-Pch1) forms a complex with the cap-methyltransferase Pcm1 and these proteins colocalise on chromatin. Ablation of Cdk9 function through chemical genetics causes growth arrest and abolishes serine 2 phosphorylation on the PolII CTD. Strikingly, depletion of Pcm1 also leads to a dramatic decrease of phospho-serine 2. Chromatin immunoprecipitations show a severe decrease of chromatin-bound Cdk9-Pch1 when Pcm1 is depleted. On the contrary, Cdk9 is not required for association of Pcm1 with chromatin. Furthermore, compromising Cdk9 activity leads to a promoter-proximal PolII stalling and sensitivity to 6-azauracil, reflecting elongation defects. The in vivo data presented here strongly support the existence of a molecular mechanism where the cap-methyltransferase recruits P-TEFb to chromatin, thereby ensuring that only properly capped transcripts are elongated.

The asymmetric distribution of the essential histidine kinase PdhS indicates a differentiation event in Brucella abortus.

Many organisms use polar localization of signalling proteins to control developmental events in response to completion of asymmetric cell division. Asymmetric division was recently reported for Brucella abortus, a class III facultative intracellular pathogen generating two sibling cells of slightly different size. Here we characterize PdhS, a cytoplasmic histidine kinase essential for B. abortus viability and homologous to the asymmetrically distributed PleC and DivJ histidine kinases from Caulobacter crescentus. PdhS is localized at the old pole of the large cell, and after division and growth, the small cell acquires PdhS at its old pole. PdhS may therefore be considered as a differentiation marker as it labels the old pole of the large cell. Moreover, PdhS colocalizes with its paired response regulator DivK. Finally, PdhS is able to localize at one pole in other alpha-proteobacteria, suggesting that a polar structure associating PdhS with one pole is conserved in these bacteria. We propose that a differentiation event takes place after the completion of cytokinesis in asymmetrically dividing alpha-proteobacteria. Altogether, these data suggest that prokaryotic differentiation may be much more widespread than expected.

hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes.

Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at ). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.

Gateway-based destination vectors for functional analyses of bacterial ORFeomes: application to the Min system in Brucella abortus.

Twenty Gateway-compatible destination vectors were constructed. The vectors comprise fluorescent and epitope fusion tags, various drug markers, and replication origins that should make them useful for exploring existing microbial ORFeomes. In an attempt to validate several of these vectors, we observed polar and oscillating localization of MinD in Brucella abortus.

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF.

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress.

Identification, cloning, and expression of the Scytalidium acidophilum XYL1 gene encoding for an acidophilic xylanase.

We cloned XYL1, a Scytalidium acidophilum gene encoding for an acidophilic family 11 xylanase. The XYL1p protein was expressed in Pichia pastoris using the pPICZalphaA expression plasmid. The secreted protein was purified by TAXI affinity column chromatography. The purified XYL1p showed an optimum activity at pH 3.2 and 56 degrees C. The Michaelis-Menten constants were determined.

Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance.

The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression.

Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe.

The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.

Glucose deprivation mediates interaction between CTDK-I and Snf1 in Saccharomyces cerevisiae.

Ctk1 is a kinase involved in transcriptional control. We show in the two-hybrid system that Ctk1 interacts with Snf1, a kinase regulating glucose-dependent genes. Co-purification experiments confirmed the two-hybrid interaction but only when cells were grown at low glucose concentrations. Deletion of Ctk1 or its associated partners, Ctk2 and Ctk3, conferred synthetic lethality with null mutants of Snf1 or Snf1-associated proteins. Northern blot analysis suggested that Ctk1 and Snf1 act together in vivo to regulate GSY2. These findings support the view that Ctk1 interacts with Snf1 in a functional module involved in the cellular response to glucose limitation.