A new use for old drugs: identifying compounds with an anti-obesity effect using a high through-put semi-automated Caenorhabditis elegans screening platform.

Obesity is one of the most common global health problems for all age groups with obese people at risk of a variety of associated health complications. Consequently, there is a need to develop new therapies that lower body fat without the side effects. However, obesity is a complex and systemic disease, so that in vitro results are not easily translatable to clinical situations. A promising way to circumnavigate these issues is to reposition already approved drugs for new treatments, enabling a more streamlined drug discovery process due to the availability of pre-existing pharmacological and toxicological datasets. Chemical libraries, such as the Prestwick Chemical Library of 1200 FDA approved drugs, are available for this purpose. We have developed a simple semi-automated whole-organism approach to screening the Prestwick Chemical Library for those compounds which reduce fat content using the model organism Caenorhabditis elegans. Our whole-organism approach to high-throughput screening identified 9 "lead" compounds that reduced fat within 2 weeks in the model. Further screening and analysis provided 4 "hit" compounds (Midodrine, Vinpocetine, Fenoprofen and Lamivudine) that showed significant promise as drugs to reduce fat levels. The effects of these candidates were found to further reduce fat content in nematodes where an nhr-49/PPAR mutation resulted in "overweight" worms. Upon unblinding the "hit" compounds, they were found to have recently been shown to have anti-obesity effects in mammalian models too. In developing a whole-animal chemical screen to identify pharmacological agents as potential anti-obesity compounds, we demonstrate how chemical libraries can be rapidly and relatively cheaply profiled for active hits. Using the nematode Caenorhabditis elegans thus enables drugs to be assessed for applicability in humans and provides a new incentive to explore drug repurposing as a feasible and efficient way to identify new anti-obesity compounds.

Overexpression of delta-12 desaturase in the yeast Schwanniomyces occidentalis enhances the production of linoleic acid.

The oleaginous yeast Schwanniomyces occidentalis was previously isolated because of its excellent suitability to convert lignocellulosic hydrolysates into triacyl glycerides: it is able to use a broad range of sugars and is able to tolerate high concentrations of lignocellulosic hydrolysate inhibitors. Compared to other oleaginous yeasts S. occidentalis however produces a low content of unsaturated fatty acids. We show here that the linoleic acid content can be significantly improved by (over)expression Δ12-desaturases derived from S. occidentalis and Fusarium moniliforme. Expression was stable for the homologous expression but decreased during heterologous expression. Both homologous and heterologous expression of mCherry-Δ12-desaturase led to a 4-fold increase in linoleic acid from 0.02 g/g biomass to 0.08 g/g biomass resulting in the production of 2.23 g/L and 2.05 g/L of linoleic acid.

DnaJ chaperones contribute to canalization.

Canalization, an intrinsic robustness of development to external (environmental) or internal (genetic) perturbations, was first proposed over half a century ago. However, whether the robustness to environmental stress (environmental canalization [EC]) and to genetic variation (genetic canalization) are underpinned by the same molecular basis remains elusive. The recent discovery of the involvement of two endoplasmic reticulum (ER)-associated DnaJ genes in developmental buffering, orthologues of which are conserved across Metazoa, indicates that the role of ER-associated DnaJ genes might be conserved across the animal kingdom. To test this, we surveyed the ER-associated DnaJ chaperones in the nematode Caenorhabditis elegans. We then quantified the phenotype, in the form of variance and mean of seam cell counts, from RNA interference knockdown of DnaJs under three different temperatures. We find that seven out of eight ER-associated DnaJs are involved in either EC or microenvironmental canalization. Moreover, we also found two DnaJ genes not specifically associated with ER (DNAJC2/dnj-11 and DNAJA2/dnj-19) were involved in canalization. Protein expression pattern showed that these DnaJs are upregulated by heat stress, yet not all of them are expressed in the seam cells. Moreover, we found that most of the buffering DnaJs also control lifespan. We therefore concluded that a number of DnaJ chaperones, not limited to those associated with the ER, are involved in canalization as a part of the complex system that underlies development.

A Novel Quality Measure and Correction Procedure for the Annotation of Microbial Translation Initiation Sites.

The identification of translation initiation sites (TISs) constitutes an important aspect of sequence-based genome analysis. An erroneous TIS annotation can impair the identification of regulatory elements and N-terminal signal peptides, and also may flaw the determination of descent, for any particular gene. We have formulated a reference-free method to score the TIS annotation quality. The method is based on a comparison of the observed and expected distribution of all TISs in a particular genome given prior gene-calling. We have assessed the TIS annotations for all available NCBI RefSeq microbial genomes and found that approximately 87% is of appropriate quality, whereas 13% needs substantial improvement. We have analyzed a number of factors that could affect TIS annotation quality such as GC-content, taxonomy, the fraction of genes with a Shine-Dalgarno sequence and the year of publication. The analysis showed that only the first factor has a clear effect. We have then formulated a straightforward Principle Component Analysis-based TIS identification strategy to self-organize and score potential TISs. The strategy is independent of reference data and a priori calculations. A representative set of 277 genomes was subjected to the analysis and we found a clear increase in TIS annotation quality for the genomes with a low quality score. The PCA-based annotation was also compared with annotation with the current tool of reference, Prodigal. The comparison for the model genome of Escherichia coli K12 showed that both methods supplement each other and that prediction agreement can be used as an indicator of a correct TIS annotation. Importantly, the data suggest that the addition of a PCA-based strategy to a Prodigal prediction can be used to 'flag' TIS annotations for re-evaluation and in addition can be used to evaluate a given annotation in case a Prodigal annotation is lacking.

CiVi: circular genome visualization with unique features to analyze sequence elements.

UNLABELLED: We have developed CiVi, a user-friendly web-based tool to create custom circular maps to aid the analysis of microbial genomes and sequence elements. Sequence related data such as gene-name, COG class, PFAM domain, GC%, and subcellular location can be comprehensively viewed. Quantitative gene-related data (e.g. expression ratios or read counts) as well as predicted sequence elements (e.g. regulatory sequences) can be uploaded and visualized. CiVi accommodates the analysis of genomic elements by allowing a visual interpretation in the context of: (i) their genome-wide distribution, (ii) provided experimental data and (iii) the local orientation and location with respect to neighboring genes. CiVi thus enables both experts and non-experts to conveniently integrate public genome data with the results of genome analyses in circular genome maps suitable for publication. CONTACT: L.Overmars@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. AVAILABILITY AND IMPLEMENTATION: CiVi is freely available at http://civi.cmbi.ru.nl.

Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

MGcV: the microbial genomic context viewer for comparative genome analysis.

BACKGROUND: Conserved gene context is used in many types of comparative genome analyses. It is used to provide leads on gene function, to guide the discovery of regulatory sequences, but also to aid in the reconstruction of metabolic networks. We present the Microbial Genomic context Viewer (MGcV), an interactive, web-based application tailored to strengthen the practice of manual comparative genome context analysis for bacteria. RESULTS: MGcV is a versatile, easy-to-use tool that renders a visualization of the genomic context of any set of selected genes, genes within a phylogenetic tree, genomic segments, or regulatory elements. It is tailored to facilitate laborious tasks such as the interactive annotation of gene function, the discovery of regulatory elements, or the sequence-based reconstruction of gene regulatory networks. We illustrate that MGcV can be used in gene function annotation by visually integrating information on prokaryotic genes, like their annotation as available from NCBI with other annotation data such as Pfam domains, sub-cellular location predictions and gene-sequence characteristics such as GC content. We also illustrate the usefulness of the interactive features that allow the graphical selection of genes to facilitate data gathering (e.g. upstream regions, ID's or annotation), in the analysis and reconstruction of transcription regulation. Moreover, putative regulatory elements and their corresponding scores or data from RNA-seq and microarray experiments can be uploaded, visualized and interpreted in (ranked-) comparative context maps. The ranked maps allow the interpretation of predicted regulatory elements and experimental data in light of each other. CONCLUSION: MGcV advances the manual comparative analysis of genes and regulatory elements by providing fast and flexible integration of gene related data combined with straightforward data retrieval. MGcV is available at http://mgcv.cmbi.ru.nl.

Comparative genome analysis of central nitrogen metabolism and its control by GlnR in the class Bacilli.

BACKGROUND: The assimilation of nitrogen in bacteria is achieved through only a few metabolic conversions between alpha-ketoglutarate, glutamate and glutamine. The enzymes that catalyze these conversions are glutamine synthetase, glutaminase, glutamate dehydrogenase and glutamine alpha-ketoglutarate aminotransferase. In low-GC Gram-positive bacteria the transcriptional control over the levels of the related enzymes is mediated by four regulators: GlnR, TnrA, GltC and CodY. We have analyzed the genomes of all species belonging to the taxonomic families Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae to determine the diversity in central nitrogen metabolism and reconstructed the regulation by GlnR. RESULTS: Although we observed a substantial difference in the extent of central nitrogen metabolism in the various species, the basic GlnR regulon was remarkably constant and appeared not affected by the presence or absence of the other three main regulators. We found a conserved regulatory association of GlnR with glutamine synthetase (glnRA operon), and the transport of ammonium (amtB-glnK) and glutamine/glutamate (i.e. via glnQHMP, glnPHQ, gltT, alsT). In addition less-conserved associations were found with, for instance, glutamate dehydrogenase in Streptococcaceae, purine catabolism and the reduction of nitrite in Bacillaceae, and aspartate/asparagine deamination in Lactobacillaceae. CONCLUSIONS: Our analyses imply GlnR-mediated regulation in constraining the import of ammonia/amino-containing compounds and the production of intracellular ammonia under conditions of high nitrogen availability. Such a role fits with the intrinsic need for tight control of ammonia levels to limit futile cycling.

Computational analysis of cysteine and methionine metabolism and its regulation in dairy starter and related bacteria.

Sulfuric volatile compounds derived from cysteine and methionine provide many dairy products with a characteristic odor and taste. To better understand and control the environmental dependencies of sulfuric volatile compound formation by the dairy starter bacteria, we have used the available genome sequence and experimental information to systematically evaluate the presence of the key enzymes and to reconstruct the general modes of transcription regulation for the corresponding genes. The genomic organization of the key genes is suggestive of a subdivision of the reaction network into five modules, where we observed distinct differences in the modular composition between the families Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, on the one hand, and the family Streptococcaceae, on the other. These differences are mirrored by the way in which transcription regulation of the genes is structured in these families. In the Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, the main shared mode of transcription regulation is methionine (Met) T-box-mediated regulation. In addition, the gene metK, encoding S-adenosylmethionine (SAM) synthetase, is controlled via the S(MK) box (SAM). The S(MK) box is also found upstream of metK in species of the family Streptococcaceae. However, the transcription control of the other modules is mediated via three different LysR-family regulators, MetR/MtaR (methionine), CmbR (O-acetyl[homo]serine), and HomR (O-acetylhomoserine). Redefinition of the associated DNA-binding motifs helped to identify/disentangle the related regulons, which appeared to perfectly match the proposed subdivision of the reaction network.

Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome.

There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.

Comparative analyses imply that the enigmatic Sigma factor 54 is a central controller of the bacterial exterior.

BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm.

Reconstruction of the regulatory network of Lactobacillus plantarum WCFS1 on basis of correlated gene expression and conserved regulatory motifs.

Gene regulatory networks can be reconstructed by combining transcriptome data from many different experiments to elucidate relations between the activity of certain transcription factors and the genes they control. To obtain insight in the regulatory network of Lactobacillus plantarum, microarray transcriptome data from more than 70 different experimental conditions were combined and the expression profiles of the transcriptional units (TUs) were compared. The TUs that displayed correlated expression were used to identify putative cis-regulatory elements by searching the upstream regions of the TUs for conserved motifs. Predicted motifs were extended and refined by searching for motifs in the upstream regions of additional TUs with correlated expression. In this way, cis-acting elements were identified for 41 regulons consisting of at least four TUs (correlation > 0.7). This set of regulons included the known regulons of CtsR and LexA, but also several novel ones encompassing genes with coherent biological functions. Visualization of the regulons and their connections revealed a highly interconnected regulatory network. This network contains several subnetworks that encompass genes of correlated biological function, such as sugar and energy metabolism, nitrogen metabolism and stress response.

Isolation of highly heat-resistant Listeria monocytogenes variants by use of a kinetic modeling-based sampling scheme.

Stable high-hydrostatic-pressure (HHP)-resistant Listeria monocytogenes LO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in their ctsR genes. For the variants whose ctsR genes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistant L. monocytogenes variants.

Clostridial spore germination versus bacilli: genome mining and current insights.

Bacilli and clostridia share the characteristic of forming metabolically inactive endospores. Spores are highly resistant to adverse environmental conditions including heat, and their ubiquitous presence in nature makes them inevitable contaminants of foods and food ingredients. Spores can germinate under favourable conditions, and the following outgrowth can lead to food spoilage and foodborne illness. Germination of spores has been best studied in Bacillus species, but the process of spore germination is less well understood in anaerobic clostridia. This paper describes a genome mining approach focusing on the genes related to spore germination of clostridia. To this end, 12 representative sequenced Bacillus genomes and 24 Clostridium genomes were analyzed for the distribution of known and putative germination-related genes and their homologues. Overall, the number of ger operons encoding germinant receptors is lower in clostridia than in bacilli, and some Clostridium species are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli. The in silico germination model constructed for clostridia was linked to recently obtained experimental data for selected germination determinants, mainly in Clostridium perfringens. Similarities and differences between germination mechanisms of bacilli and clostridia will be discussed.

Novel sigmaB regulation modules of Gram-positive bacteria involve the use of complex hybrid histidine kinases.

A common bacterial strategy to cope with stressful conditions is the activation of alternative sigma factors that control specific regulons enabling targeted responses. In the human pathogen Bacillus cereus, activation of the major stress-responsive sigma factor σ(B) is controlled by a signalling route that involves the multi-sensor hybrid histidine kinase RsbK. RsbK-type kinases are not restricted to the B. cereus group, but occur in a wide variety of other bacterial species, including members of the the low-GC Gram-positive genera Geobacillus and Paenibacillus as well as the high-GC actinobacteria. Genome context and protein sequence analyses of 118 RsbK homologues revealed extreme variability in N-terminal sensory as well as C-terminal regulatory domains and suggested that RsbK-type kinases are subject to complex fine-tuning systems, including sensitization and desensitization via methylation and demethylation within the helical domain preceding the H-box. The RsbK-mediated stress-responsive sigma factor activation mechanism that has evolved in B. cereus and the other species differs markedly from the extensively studied and highly conserved RsbRST-mediated σ(B) activation route found in Bacillus subtilis and other low-GC Gram-positive bacteria. Implications for future research on sigma factor control mechanisms are presented and current knowledge gaps are briefly discussed.

Complete genome sequence of the probiotic Lactobacillus casei strain BL23.

The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus.

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1(Lp) and ram2(Lp)) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA(La)). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1(Lp), Ram2(Lp), and RamA(La) were able to efficiently hydrolyze rutin and other rutinosides, while RamA(La) was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1(Lp) and RamA(La) enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1(Lp) for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.

The identification of response regulator-specific binding sites reveals new roles of two-component systems in Bacillus cereus and closely related low-GC Gram-positives.

In bacteria, environmental challenges are often translated into a transcriptional response via the cognate response regulators (RRs) of specialized two-component systems (TCSs). A phylogenetic footprinting/shadowing approach was designed and used to identify many novel RR-specific operators for species of the Bacillus cereus group and related Gram-positives. Analysis of the operator sequences revealed characteristic traits for each RR subfamily. For instance, operators related to the largest subfamily (OmpR) typically consisted of direct repeats (e.g. TTAAGA-N5-TTAAGA), whereas operators related to the second largest family (NarL) consisted of inverted repeats (e.g. ATGACA-N2-TGTCAT). This difference indicates a fundamentally different organization of the bound RR dimers between the two subfamilies. Moreover, the identification of the specific operator motifs allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. Mostly, these regulons comprised genes encoding transport systems, suggesting a direct coupling of stimulus perception to the transport of target compounds. New biological roles could be attributed to various TCSs, including roles in cytochrome c biogenesis (HssRS), transport of carbohydrates, peptides and/or amino acids (YkoGH, LytSR), and resistance to toxic ions (LiaSR), antimicrobial peptides (BceRS) and beta-lactam antibiotics (BacRS, YcbLM). As more and more bacterial genome sequences are becoming available, the use of comparative analyses such as the approach applied in this study will further increase our knowledge of bacterial signal transduction mechanisms and provide directions for the assessment of their role in bacterial performance and survival strategies.

An in silico analysis of T-box regulated genes and T-box evolution in prokaryotes, with emphasis on prediction of substrate specificity of transporters.

BACKGROUND: T-box anti-termination is an elegant and sensitive mechanism by which many bacteria maintain constant levels of amino acid-charged tRNAs. The amino acid specificity of the regulatory element is related to a so-called specifier codon and can in principle be used to guide the functional annotation of the genes controlled via the T-box anti-termination mechanism. RESULTS: Hidden Markov Models were defined to search the T-box regulatory element and were applied to all completed prokaryotic genomes. The vast majority of the genes found downstream of the retrieved elements encoded functionalities related to transport and synthesis of amino acids and the charging of tRNA. This is completely in line with findings reported in literature and with the proposed biological role of the regulatory element. For several species, the functional annotation of a large number of genes encoding proteins involved in amino acid transport could be improved significantly on basis of the amino acid specificity of the identified T-boxes. In addition, these annotations could be extrapolated to a larger number of orthologous systems in other species. Analysis of T-box distribution confirmed that the element is restricted predominantly to species of the phylum Firmicutes. Furthermore, it appeared that the distribution was highly species specific and that in the case of amino acid transport some boxes seemed to "pop-up" only recently. CONCLUSION: We have demonstrated that the identification of the molecular specificity of a regulatory element can be of great help in solving notoriously difficult annotation issues, e.g. by defining the substrate specificity of genes encoding amino acid transporters on basis of the amino acid specificity of the regulatory T-box. Furthermore, our analysis of the species-dependency of the occurrence of specific T-boxes indicated that these regulatory elements propagate in a semi-independent way from the genes that they control.