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Background: The SOS response including two main proteins LexA and RecA, maintains the integrity of bacterial genomes after DNA damage due to metabolic or environmental assaults. Additionally, derepression of LexA-regulated genes can result in mutations, genetic exchange and expression of virulence factors. Here we describe the first comprehensive description of the in silico LexA regulon in Clostridium difficile, an important human pathogen. Results: We grouped thirty C. difficile strains from different ribotypes and toxinotypes into three clusters according to lexA gene/protein variability. We applied in silico analysis coupled to surface plasmon resonance spectroscopy (SPR) and determined 16 LexA binding sites in C. difficile. Our data indicate that strains within the cluster, as defined by LexA variability, harbour several specific LexA regulon genes. In addition to core SOS genes: lexA, recA, ruvCA and uvrBA, we identified a LexA binding site on the pathogenicity locus (PaLoc) and in the putative promoter region of several genes involved in housekeeping, sporulation and antibiotic resistance. Conclusions: Results presented here suggest that in C. difficile LexA is not merely a regulator of the DNA damage response genes but also controls the expression of dozen genes involved in various other biological functions. Our in vitro results indicate that in C. difficile inactivation of LexA repressor depends on repressor`s dissociation from the operators. We report that the repressor`s dissociation rates from operators differentiate, thus the determined LexA-DNA dissociation constants imply on the timing of SOS gene expression in C. difficile.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Effect of SHAM and KCN on fungal growth and sporulation. (PDF 252 kb)

Maximum likelihood rooted phylogeny of the FCY-like transporters of the aspergilli and some penicillia. (PDF 324 kb)

Comparative growth profiling of the aspergilli and selected other fungi. (PDF 8.65 kb)

Overview of number of putative orthologs/paralogs in the compared fungal species related to primary carbon metabolism. (PDF 237 kb)

Expression of mating and pheromone-signalling pathway genes in representative asexual aspergilli. (PDF 488 kb)

Extended analysis of stress tolerance of the aspergilli. (PDF 706 kb)

Maximal likelihood phylogeny of MASC1/RID1 orthologs. (PDF 115 kb)

Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.

β2-glycoprotein I (β2GPI) is a major autoantigen of autoimmune thrombophilia, known as the antiphospholipid syndrome. The exact mechanism underlying the β2GPI's involvement in the disease is not fully elucidated, as it is not its physiological role. We used random phage peptide library to identify sequences binding to β2GPI. Obtained K(L/V)WX(I/L/V)P motif, primarily designated as target unrelated, was confirmed as the selective binder of β2GPI. Based on this motif we confirmed the previously suggested role of polar residues in β2GPI interactions, and identified some already known and some new putative β2GPI binding proteins. The latter can help to further elucidate β2GPI's (patho)physiological role.

Aminoacyl-tRNA synthetases (aaRS) support translational process by attaching appropriate amino acids to cognate tRNAs. Charged tRNA molecules reach ribosome and the amino acid can be transfered onto a growing peptide, according to the genetic code. It is well known that aaRSs can be involved in diverse cellular functions beyond translation independently or by forming protein- protein interactions. Understanding these non- canonical functions opens new perspectives in functional proteomics. However, the studies of plant aaRSs are scarce. Therefore our recent findings on interaction of BEN1, protein involved in metabolism of brassinosteroid hormones, and cytosolic seryl-tRNA synthetase (SerRS) from plant Arabidopsis thaliana broadens our knowledge of novel non-translational functions of plant aaRSs. We detected BEN1:SerRS interaction in vivo using Y2H screen and confirmed it in vitro using SPR method, also determining the dissociation constant of the protein complex. To pinpoint regions responsible for protein-protein interaction truncated variants of both SerRS and BEN1 protein were prepared and analyzed using microscale thermophoresis assay, that also enables Kd determination. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension was similar to Kd of the SerRS:BEN1 complex indicating that basic C-terminal extension does not participate in interaction. Furthermore, N-terminal tRNA binding domain of SerRS did not form the complex with BEN1. Therefore we conclude that interaction involves the central part of SerRS that contains globular catalytic domain. Furthermore, using reverse transcription- quantitative polymerase chain reaction we investigated possible correlation of these two proteins on the level of gene expression in wild type plants, but also in transgenic plants overexpressing SerRS. We observed slightly lower expression of BEN1 gene in transgenic plants compared to wild type. Combining insights from both gene and protein level allowed us to define interaction surfaces and propose functional importance of SerRS:BEN1 assembly.

Aminoacyl-tRNA synthetases (aaRS) are well known to interact with other cellular proteins. In the archaeon Methanothermobacter thermautotrophicus a complex is formed by the transient gathering of the arginyl- and seryl-tRNA synthetase (ArgRS and SerRS). Pull-down and surface plasmon resonance experiments with truncated ArgRS variants showed that the N-terminal part of ArgRS serves as the recognition platform for SerRS, with emphasis on the elements involved in tRNAArg recognition. The shortened variants Δ89-ArgRS and ΔNtot-ArgRS show decreased ability for cognate tRNA binding and charging. Regarding the possible effect that reaction precursors may have on the stability of ArgRS:SerRS complex, it is shown that the SerRS substrates exert no influence. Furthermore, tRNASer is able to participate in ternary [SerRS:tRNASer]:ArgRS complex formation with a stability comparable to that of the binary SerRS:ArgRS assembly. In contrast, interaction of these proteins is susceptible to the addition of fully modified tRNAArg transcript which ultimately leads to dissociation of the complex. Overexpression of tRNAArg in E. coli cells results with the production of three distinct molecular species which differ in the amount of added posttranscriptional modifications. Arginyl-tRNA synthetase investigated in this work aminoacylates such substrates with comparable efficacy. Interestingly, SerRS is able to inhibit arginylation of poorer substrates, i.e., less modified tRNAArg molecules. Elimination of the partly modified tRNAs from the translation process shows how this protein complex may act as a safeguarding switch improving decoding accuracy.

It is well known that aaRSs can be involved in diverse cellular functions beyond translation independently or by forming protein-protein interactions. However, the studies of plant aaRS assemblies are scarce. We used high- throughput interactome technologies to identify potential protein interactors of Arabidopsis thaliana SerRS. We generated transgenic plants expressing SerRS-TAP and subsequent TAP-MS analysis revealed several potential interactors, including glutathione S- transferase PM24 and aluminium induced protein AT4g27450. Yeast two hybrid screen indicated interaction with BEN1, protein involved in metabolism of steroidal plant hormones brassinosteroids. These plant growth- promoting hormones regulate a variety of physiological processes crucial for normal plant growth and development and are also involved in plant stress response. The SerRS:BEN1 interaction was confirmed using surface plasmon resonance and microscale thermophoresis (MST), that enable Kd determination. To pinpoint regions responsible for interaction, truncated variants of both SerRS and BEN1 proteins were prepared and analyzed using MST. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension was similar to Kd of the SerRS:BEN1 complex indicating that basic C- terminus does not participate in interaction. N-terminal tRNA binding domain of SerRS did not form the complex with BEN1. Deletion of N- terminal extension of BEN1 containing six acidic amino acids disrupted the interaction. Therefore we conclude that interaction involves SerRS globular catalytic domain and acidic N- terminus of BEN1. Furthermore, using RT-qPCR we investigated possible correlation of these two proteins on the level of gene expression. We observed slightly lower expression of BEN1 gene in transgenic plants overexpressing SerRS compared to wild type. To test whether SerRS regulates BEN1 expression in nucleus we determined its localization using GFP- localization experiments and immunoblot analysis. Our data show that SerRS is exclusively localized in cytosol, despite its several putative nuclear localization signals. To investigate the role of SerRS in stress, we examined growth of transgenic seedlings overexpressing SerRS on growth media that induce ionic stress, osmotic stress and stress imposed by heavy metals. Our results show that transgenic seeds germinate earlier and that in most cases transgenic seedlings grow better on stress media compared to wild type seedlings. This indicates that enhanced expression of SerRS plays a role in the response of plant to various stress conditions. Considering that brassinosteroids are also involved in plant stress response we propose functional importance of SerRS:BEN1 assembly.

Aminoacyl-tRNA synthetases (aaRS) attach appropriate amino acids to cognate tRNAs ensuring efficient protein biosynthesis. Their involvement in diverse celullar functions beyond translation opens broad new perspectives in functional proteomics, especially in the field of plant aaRSs which is fairly uninvestigated. To shed light on non-canonical functions of cytosolic seryl-tRNA synthetase (SerRS) from plant Arabidopsis thaliana we conducted yeast two hybrid screen (Y2H) which revealed metabolic protein BEN1 as a highly promising interacting partner. Interaction was further biophysicaly analyzed in vitro using isothermal calorimetry titration (ITC), pull- down, surface plasmon resonance (SPR) and microscale thermophoresis method (MST). Due to the nature of interaction and sensitivity of applied methods we were not able to retrieve positive results using pull-down assay and ITC, but SPR and MST gave us postive confirmation of interaction and information about dissociation constant (Kd constant obtained using SPR = 1, 03 × 10-6 (± 1, 67 × 10-7) mol dm-3, in good agreement with Kd obtained using MST = 4, 45 × 10-7 (± 1, 40 × 10-7) mol dm-3). To determine interaction surfaces and pinpoint regions responsible for SerRS and BEN1 interaction, truncated variants of both SerRS and BEN1 proteins were prepared and protein interactions were analyzed using MST. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension (with or without (His)6 tag) was similar to Kd of the SerRS:BEN1 complex indicating that SerRS basic C- terminal extension does not have any influence on interaction. Furthermore, isolated N-terminal domain of SerRS did not form the complex with BEN1. Taking into account observed data, we concluded that interaction between SerRS and BEN1 involves the central part of SerRS that contains globular catalytic domain. Determining interaction domain of BEN1 was not possible because of aggregation problems in MST assay when using truncated BEN1 version, so this part of investigation is yet to be done. Knowing precise interaction contacts will allow us to understand and propose functional importance of SerRS:BEN1 assembly in plant organism.

Aminoacyl-tRNA synthetases (aaRS) play significant role in translation process by binding amino acids to their cognate tRNAs. Once the tRNA is charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to the genetic code. Beyond translation, these enzymes can be involved in diverse cellular functions. Characterization of these non-canonical functions broadens our knowledge in functional proteomics. The studies of aaRS assemblies in plants are scarce, therefore our main scientific goals were to determine and characterize potential protein- protein interacting partners of cytosolic seryl- tRNA synthetase (SerRS) from plant Arabidopsis thaliana. We conducted yeast-two hybrid (Y2H) screen on cDNA libraries followed by DNA sequencing, as well as tandem affinity purification combined with mass spectrometry analysis (TAP-MS). Among several SerRS potential interacting partners revealed by Y2H screen, BEN1, protein potentially involved in metabolism of brassinosteroid hormones was the most promising interacting partner. Interaction of BEN1 and SerRS was also analyzed in vitro using isothermal calorimetry titration (ITC), pull-down and surface plasmon resonance method (SPR). Probably due to the nature of interaction we were not able to retrieve positive results using pull-down assay and ITC, but SPR gave us positive confirmation and information about dissociation constant. To pinpoint regions responsible for protein- protein interaction we prepared shortened variants of both SerRS and BEN1 proteins which will be further subjected to biophysical analysis. Biophysical determination of possible interactions between SerRS and BEN1 variants will give us insight in additional cell functions and physiology of both SerRS and BEN1 proteins. TAP-MS analysis of transgenic plant overexpressing SerRS-TAP construct identified several SerRS potential interacting partners yet to be confirmed in vitro. Revealing plant SerRS interactome has great importance in shedding light on novel functions of SerRS beyond translation.

Aminoacyl-tRNA synthetases (AARS) are essential cellular enzymes that play significant role in protein synthesis by charging amino acids to their cognate tRNAs. In recent years more evidence appeared showing involvement of these enzymes in diverse cellular functions beyond translation. Characterization of these non-canonical functions broadens our knowledge in functional proteomics. The studies of aaRS assemblies in plants are scarce. Our main research goals were to determine potential protein interacting partners of seryl- tRNA synthetase (SerRS) in plant cell, its potential nuclear localization and kinetic characterization. In search for SerRS interacting protein partners in plant Arabidopsis thaliana, L. we conducted yeast-two hybrid (Y2H) screen on cDNA libraries and tandem affinity purification (TAP) in plant. Potential interacting partners were identified either by DNA sequencing (Y2H) or mass spectrometry (TAP). BEN1, protein involved in metabolism of brassinosteroid hormones, was identified as the most promising interacting partner. Interaction of BEN1 and SerRS was analyzed in vitro using isothermal calorimetry titration (ITC), pull-down and surface plasmon resonance method (SPR). Probably due to the nature of interaction we were not able to retrieve positive results using pull-down assay and ITC, but SPR gave us positive confirmation and information about dissociation constant. To determine regions responsible for protein-protein interaction we planned to prepare shortened variants of both SerRS and BEN1 proteins and use them in biophysical analysis. So far we managed to construct, successfully express and purify BEN1 variant lacking 37 amino acids long polar region at the N-terminus and SerRS variant lacking hydrophilic region at C-terminus. Although we successfully cloned other truncated variants of SerRS protein, thus far their expression yielded inclusion bodies. Biophysical determination of possible interactions between SerRS and BEN1 variants will give us insight in additional cell functions and physiology of both SerRS and BEN1 proteins.