ALBERT

Description: Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information. Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

Description: Data on biological mechanisms of aging are mostly obtained from cross-sectional study designs. An inherent disadvantage of this design is that inter-individual differences can mask small but biologically significant age-dependent changes. A serially sampled design (same individual at different time points) would overcome this problem but is often limited by the relatively small numbers of available paired samples and the statistics being used. To overcome these limitations, we have developed a new vector-based approach, termed three-component analysis, which incorporates temporal distance, signal intensity and variance into one single score for gene ranking and is combined with gene set enrichment analysis. We tested our method on a unique age-based sample set of human skin fibroblasts and combined genome-wide transcription, DNA methylation and histone methylation (H3K4me3 and H3K27me3) data. Importantly, our method can now for the first time demonstrate a clear age-dependent decrease in expression of genes coding for proteins involved in translation and ribosome function. Using analogies with data from lower organisms, we propose a model where age-dependent down-regulation of protein translation-related components contributes to extend human lifespan.

Description: A major challenge in the field of shotgun metagenomics is the accurate identification of organisms present within a microbial community, based on classification of short sequence reads. Though existing microbial community profiling methods have attempted to rapidly classify the millions of reads output from modern sequencers, the combination of incomplete databases, similarity among otherwise divergent genomes, errors and biases in sequencing technologies, and the large volumes of sequencing data required for metagenome sequencing has led to unacceptably high false discovery rates (FDR). Here, we present the application of a novel, gene-independent and signature-based metagenomic taxonomic profiling method with significantly and consistently smaller FDR than any other available method. Our algorithm circumvents false positives using a series of non-redundant signature databases and examines G enomic O rigins T hrough T axonomic CHA llenge (GOTTCHA). GOTTCHA was tested and validated on 20 synthetic and mock datasets ranging in community composition and complexity, was applied successfully to data generated from spiked environmental and clinical samples, and robustly demonstrates superior performance compared with other available tools.

Description: The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE017354.1) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

Description: Thermotolerance of the fungus Fomes sp. EUM1 was evaluated in solid state fermentation (SSF). This thermotolerant strain improved both hyphal invasiveness (38%) and length (17%) in adverse thermal conditions exceeding 30°C and to a maximum of 40°C. In contrast, hyphal branching decreased by 46% at 45°C. The production of cellulases over corn stover increased 1.6-fold in 30°C culture conditions, xylanases increased 2.8-fold at 40°C, while laccase production improved 2.7-fold at 35°C. Maximum production of lignocellulolytic enzymes was obtained at elevated temperatures in shorter fermentation times (8–6 days), although the proteases appeared as a thermal stress response associated with a drop in lignocellulolytic activities. Novel and multiple isoenzymes of xylanase (four bands) and cellulase (six bands) were secreted in the range of 20–150 kDa during growth in adverse temperature conditions. However, only a single laccase isoenzyme (46 kDa) was detected. This is the first report describing the advantages of a thermotolerant white-rot fungus in SSF. These results have important implications for large-scale SSF, where effects of metabolic heat are detrimental to growth and enzyme production, which are severely affected by the formation of high temperature gradients.

Description: The rhizobacterium Serratia plymuthica 4Rx13 emits the novel and unique volatile sodorifen (C 16 H 26 ), which has a polymethylated bicyclic structure. Transcriptome analysis revealed that gene SOD_c20750 (annotated as terpene cyclase) is involved in the biosynthesis of sodorifen. Here we show that this gene is located in a small cluster of four genes ( SOD_c20750 – SOD_c20780 ), and the analysis of the knockout mutants demonstrated that SOD_c20760 (annotated as methyltransferase) and SOD_c20780 (annotated as isopentenyl pyrophosphate (IPP) isomerase) are needed for the biosynthesis of sodorifen, while a sodorifen-negative phenotype was not achieved with the SOD_c20770 (annotated as deoxy-xylulose-5-phosphate (DOXP) synthase) mutant. Altogether, the function of this new gene cluster was assigned to the biosynthesis of this structurally unusual volatile compound sodorifen.

Description: Among staphylococci Staphylococcus saprophyticus is the only species that is typically uropathogenic and an important cause of urinary tract infections in young women. The amino acid D-serine occurs in relatively high concentrations in human urine and has a bacteriostatic or toxic effect on many bacteria. In uropathogenic Escherichia coli and S. saprophyticus , the amino acid regulates the expression of virulence factors and can be used as a nutrient. The ability of uropathogens to respond to or to metabolize D-serine has been suggested as a factor that enables colonization of the urinary tract. Until now nothing is known about D-serine transport in S.   saprophyticus . We generated mutants of putative transporter genes in S.   saprophyticus 7108 that show homology to the D-serine transporter cyc A of E. coli and tested them in a D-serine depletion assay to analyze the D-serine uptake rate of the cells. The mutant of SPP1070 showed a strong decrease in D-serine uptake. Therefore, SSP1070 was identified as a major D-serine transporter in S. saprophyticus 7108 and was named D-serine transporter A (DstA). D-serine caused a prolonged lag phase of S. saprophyticus in a chemically defined medium. This negative effect was dependent on the presence of DstA.

Description: Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic  Streptococcus mutans  bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of  S. mutans . The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout  S. mutans  UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted  Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout  S. mutans  strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted  E. coli  cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in  S. mutans .

Description: Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are bacteria under investigation for production of biofuels from plant biomass. Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at high yield (〉90% of theoretical) and titer (〉70 g/l). Efforts to engineer C. thermocellum have not, to date, been as successful, and efforts are underway to transfer the ethanol production pathway from T. saccharolyticum to C. thermocellum . One potential challenge in transferring metabolic pathways is the possibility of incompatible levels of nicotinamide cofactors. These cofactors (NAD + , NADH, NADP + and NADPH) and their oxidation state are important in the context of microbial redox metabolism. In this study we directly measured the concentrations and reduced oxidized ratios of these cofactors in a number of strains of C. thermocellum and T. saccharolyticum by using acid/base extraction and enzymatic assays. We found that cofactor ratios are maintained in a fairly narrow range, regardless of the metabolic network modifications considered. We have found that the ratios are similar in both organisms, which is a relevant observation in the context of transferring the T. saccharolyticum ethanol production pathway to C. thermocellum .

Description: Chromosome-long haplotyping of human genomes is important to identify genetic variants with differing gene expression, in human evolution studies, clinical diagnosis, and other biological and medical fields. Although several methods have realized haplotyping based on sequencing technologies or population statistics, accuracy and cost are factors that prohibit their wide use. Borrowing ideas from group testing theories, we proposed a clone-based haplotyping method by overlapping pool sequencing. The clones from a single individual were pooled combinatorially and then sequenced. According to the distinct pooling pattern for each clone in the overlapping pool sequencing, alleles for the recovered variants could be assigned to their original clones precisely. Subsequently, the clone sequences could be reconstructed by linking these alleles accordingly and assembling them into haplotypes with high accuracy. To verify the utility of our method, we constructed 130 110 clones in silico for the individual NA12878 and simulated the pooling and sequencing process. Ultimately, 99.9% of variants on chromosome 1 that were covered by clones from both parental chromosomes were recovered correctly, and 112 haplotype contigs were assembled with an N50 length of 3.4 Mb and no switch errors. A comparison with current clone-based haplotyping methods indicated our method was more accurate.

Description: Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these ‘repressed transcripts’ have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.

Description: DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine–guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.

Description: The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species 〉15 kb and the remaining 〉10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.

Description: Chromatin modifiers and histone modifications are components of a chromatin-signaling network involved in transcription and its regulation. The interactions between chromatin modifiers and histone modifications are often unknown, are based on the analysis of few genes or are studied in vitro . Here, we apply computational methods to recover interactions between chromatin modifiers and histone modifications from genome-wide ChIP-Seq data. These interactions provide a high-confidence backbone of the chromatin-signaling network. Many recovered interactions have literature support; others provide hypotheses about yet unknown interactions. We experimentally verified two of these predicted interactions, leading to a link between H4K20me1 and members of the Polycomb Repressive Complexes 1 and 2. Our results suggest that our computationally derived interactions are likely to lead to novel biological insights required to establish the connectivity of the chromatin-signaling network involved in transcription and its regulation.

Description: With read lengths of currently up to 2 x 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%.

Description: RNA-seq is a sensitive and accurate technique to compare steady-state levels of RNA between different cellular states. However, as it does not provide an account of transcriptional activity per se , other technologies are needed to more precisely determine acute transcriptional responses. Here, we have developed an easy, sensitive and accurate novel computational method, iRNA-seq , for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data. Comparison of the results derived from iRNA-seq analyses with parallel results derived using current methods for genome-wide determination of transcriptional activity, i.e. global run-on (GRO)-seq and RNA polymerase II (RNAPII) ChIP-seq, demonstrate that iRNA-seq provides similar results in terms of number of regulated genes and their fold change. However, unlike the current methods that are all very labor-intensive and demanding in terms of sample material and technologies, iRNA-seq is cheap and easy and requires very little sample material. In conclusion, iRNA-seq offers an attractive novel alternative to current methods for determination of changes in transcriptional activity at a genome-wide level.

Description: Identifying large-scale structural variation in cancer genomes continues to be a challenge to researchers. Current methods rely on genome alignments based on a reference that can be a poor fit to highly variant and complex tumor genomes. To address this challenge we developed a method that uses available breakpoint information to generate models of structural variations. We use these models as references to align previously unmapped and discordant reads from a genome. By using these models to align unmapped reads, we show that our method can help to identify large-scale variations that have been previously missed.

Description: Cadmium is a widespread environmental pollutant and poses some potential risks to human health. However, the signaling events controlling cadmium toxicity are not fully understood. In this study, we examined the effect of cadmium chloride on cell viability and the intracellular nitric oxide (NO) level in yeast cells. The results showed that exposure of yeast cells to cadmium (0–100 μM) could induce cell killing with significantly increased intracellular NO levels. Morphological analysis of the nuclei with 4 ' ,6-diamidino-2-phenylindole staining and DNA strand breaks analysis showed that cadmium at 50 μM can induce cell apoptosis in yeast cells. Treatment of yeast cells with cadmium (50 μM) and the nitric oxide scavenger c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-1-oxyl-3-oxide; 0.2 mM] showed that c-PTIO attenuated the cadmium-induced cell killing. Our findings indicated that cadmium-induced yeast cell killing is mediated by a directly increased intracellular NO level.

Description: Effective uptake of fermentable substrates is a fundamentally important aspect of any fermentation process. The solventogenic bacterium Clostridium beijerinckii is noted for its ability to ferment a wide range of carbohydrates, yet few of its sugar transport systems have been characterized. In common with other anaerobes, C. beijerinckii shows a marked dependence on the PEP-dependent phosphotransferase system (PTS) for sugar accumulation. In this study, the gene cbe0751 encoding the sugar-specific domains of a phosphotransferase belonging to the glucose family was cloned into an Escherichia coli strain lacking the ability to take up and phosphorylate glucose. Transformants gained ability to ferment glucose, and also mannose, and further analysis of a selected transformant demonstrated that it could take up and phosphorylate glucose, confirming that cbe0751 encodes a glucose PTS which also recognizes mannose as a substrate. RT-PCR analysis showed that cbe0751 was expressed in cultures grown on both substrates, but also to varying extents during growth on some other carbon sources. Although analogue inhibition studies suggested that Cbe0751 is not the only glucose PTS in C. beijerinckii , this system should nevertheless be regarded as a potential target for metabolic engineering to generate a strain showing improved sugar fermentation properties.

Description: Any given human individual carries multiple genetic variants that disrupt protein-coding genes, through structural variation, as well as nucleotide variants and indels. Predicting the phenotypic consequences of a gene disruption remains a significant challenge. Current approaches employ information from a range of biological networks to predict which human genes are haploinsufficient (meaning two copies are required for normal function) or essential (meaning at least one copy is required for viability). Using recently available study gene sets, we show that these approaches are strongly biased towards providing accurate predictions for well-studied genes. By contrast, we derive a haploinsufficiency score from a combination of unbiased large-scale high-throughput datasets, including gene co-expression and genetic variation in over 6000 human exomes. Our approach provides a haploinsufficiency prediction for over twice as many genes currently unassociated with papers listed in Pubmed as three commonly-used approaches, and outperforms these approaches for predicting haploinsufficiency for less-studied genes. We also show that fine-tuning the predictor on a set of well-studied ‘gold standard’ haploinsufficient genes does not improve the prediction for less-studied genes. This new score can readily be used to prioritize gene disruptions resulting from any genetic variant, including copy number variants, indels and single-nucleotide variants.

Description: Meta-analysis of gene expression has enabled numerous insights into biological systems, but current methods have several limitations. We developed a method to perform a meta-analysis using the elastic net, a powerful and versatile approach for classification and regression. To demonstrate the utility of our method, we conducted a meta-analysis of lung cancer gene expression based on publicly available data. Using 629 samples from five data sets, we trained a multinomial classifier to distinguish between four lung cancer subtypes. Our meta-analysis-derived classifier included 58 genes and achieved 91% accuracy on leave-one-study-out cross-validation and on three independent data sets. Our method makes meta-analysis of gene expression more systematic and expands the range of questions that a meta-analysis can be used to address. As the amount of publicly available gene expression data continues to grow, our method will be an effective tool to help distill these data into knowledge.

Description: Heavy metals are dense chemicals with dual biological role as micronutrients and intoxicants. A few hypersaline environmental systems are naturally enriched with heavy metals, while most metal-contaminated sites are a consequence of human activities. Numerous halotolerant and moderately halophilic Bacteria possess metal tolerance, whereas a few archaeal counterparts share similar features. The main mechanisms underlying heavy metal resistance in halophilic Bacteria and Archaea include extracellular metal sequestration by biopolymers, metal efflux mediated by specific transporters and enzymatic detoxification. Biotransformation of metals by halophiles has implications both for trace metal turnover in natural saline ecosystems and for development of novel bioremediation strategies.

Description: We screened for a gene that inhibits streptomycin production in Streptomyces griseus when it is introduced on a high-copy-number plasmid pIJ702, and obtained a plasmid pKM545. The introduction of pKM545 abolished streptomycin production on all media tested including YMP-sugar and Nutrient broth. S1 protection analysis demonstrated that the introduction of this plasmid downregulated the transcriptional activity of the promoter preceding strR , the pathway-specific transcriptional regulator for streptomycin biosynthesis. The 2.8-kb Bam HI fragment cloned onto pKM545 contained two coding sequences SGR_5442 and 5443. These coding sequences and the two downstream ones (SGR_5444 and 5445) constituted a possible operon structure designated to be rspABCD (regulation of streptomycin production). RspB and RspC exhibited a marked similarity with an ATP-binding domain and a membrane-associating domain of an ABC-2 type transporter, respectively, suggesting that the Rsp proteins comprise a membrane exporter. The gene cluster consisting of the rsp operon and the upstream divergent small coding sequence (SGR_5441) was widely distributed to Streptomyces genome. An rspB mutant of S. griseus produced 3-fold streptomycin of the parental strain in YMP liquid medium. The evidence implies that the Rsp translocator is involved in the export of a substance that specifies the expression level of streptomycin biosynthesis genes in S. griseus .

Description: We previously demonstrated that in Streptomyces coelicolor two-component system AfsQ1/Q2 activates the production of the yellow-colored coelimycin P2 (also named as yCPK) on glutamate-supplemented minimal medium, and the response regulator AfsQ1 could specifically bind to the intergenic region between two structural genes, cpkA and cpkD . Here, a more in-depth investigation was performed to elucidate the mechanism underlying the role of AfsQ1/Q2 in regulating coelimycin P2 biosynthesis. Deletion of afsQ1/Q2 resulted in markedly decreased expression of the whole coelimycin P2 biosynthetic gene cluster. Electrophoretic mobility shift assays revealed that AfsQ1 bound only to the target site identified previously, but not to any other promoters in the gene cluster. Mutations of AfsQ1-binding motif only resulted in drastically reduced transcription of the cpkA/B/C operon (encoding three type I polyketide synthases) and intriguingly, led to enhanced expression of some coelimcyin P2 genes, particularly accA1 and scF . These results suggested the direct role of AfsQ1/Q2 in regulating coelimycin production, which is directly mediated by the structural genes, but not the cluster-situated regulatory genes, and also implied that other unknown mechanisms may be involved in AfsQ1/Q2-mediated regulation of coelimycin P2 biosynthesis.

Description: Sulfate-reducing bacteria (SRBs) gain their energy by coupling the oxidation of organic substrate to the reduction of sulfate to sulfide. Several SRBs are able to use alternative terminal electron acceptors to sulfate such as nitrate. Nitrate-reducing SRBs have been isolated from a diverse range of environments. In order to be able to understand the significance of nitrate reduction in SRBs, we need to examine the ecology and physiology of the nitrate-reducing SRB isolates.

Description: The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis . The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE–6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes.

Description: The effect of fructose 1,6-bisphosphate (Fru 1,6-P 2 ) on the regulatory enzymes of pentose phosphate pathway of Escherichia coli was examined. Fru 1,6-P 2 inhibited E. coli transaldolase (EC 2.2.1.2) competitively against fructose 6-phosphate and uncompetitively against erythrose 4-phosphate, whereas Fru 1,6-P 2 did not affect glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Kinetic results can be explained by assuming that transaldolase has two kinds of binding sites for Fru 1,6-P 2 : a competitive binding site for fructose 6-phosphate and a second binding site on the enzyme-erythrose 4-phosphate complex. Fru 1,6-P 2 increased resulting from the stimulation of glycolysis, can inhibit transaldolase and further participates in the elevation of the concentration of ribose 5-phosphate that can be preferentially utilized for anabolic reaction in exponential phase of E. coli .

Description: The Hdr (heterodisulfide reductase)-like enzyme is predicted, from gene transcript profiling experiments previously published, to be essential in oxidative sulfur metabolism in a number of bacteria and archaea. Nevertheless, no biochemical and physicochemical data are available so far about this enzyme. Genes coding for it were identified in Aquifex aeolicus , a Gram-negative, hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium that uses inorganic sulfur compounds as electron donor to grow. We provide biochemical evidence that this Hdr-like enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or elemental sulfur). We demonstrate, by immunolocalization and cell fractionation, that Hdr-like enzyme is associated, presumably monotopically, with the membrane fraction. We show by co-immunoprecipitation assay or partial purification, that the Hdr proteins form a stable complex composed of at least five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present in two forms of high molecular mass on native gel (~240 and 450 kDa). These studies allow us to propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus , with Hdr predicted to generate sulfite.

Description: Geobacter sulfurreducens is an anaerobic soil bacterium that is involved in biogeochemical cycles of elements such as Fe and Mn. Although significant progress has been made in the understanding of the electron transfer processes in G. sulfurreducens , little is known about the regulatory mechanisms involved in their control. To expand the study of gene regulation in G. sulfurreducens , we carried out a genome-wide identification of transcription start sites (TSS) by 5'RACE and by deep RNA sequencing of primary mRNAs in two growth conditions. TSSs were identified along G. sulfurreducens genome and over 50% of them were located in the upstream region of the associated gene, and in some cases we detected genes with more than one TSS. Our global mapping of TSSs contributes with valuable information, which is needed for the study of transcript structure and transcription regulation signals and can ultimately contribute to the understanding of transcription initiation phenomena in G. sulfurreducens .

Description: Advanced sequencing technologies have generated a plethora of data for many chromatin marks in multiple tissues and cell types, yet there is lack of a generalized tool for optimal utility of those data. A major challenge is to quantitatively model the epigenetic dynamics across both the genome and many cell types for understanding their impacts on differential gene regulation and disease. We introduce IDEAS, an i ntegrative and d iscriminative e pigenome a nnotation s ystem, for jointly characterizing epigenetic landscapes in many cell types and detecting differential regulatory regions. A key distinction between our method and existing state-of-the-art algorithms is that IDEAS integrates epigenomes of many cell types simultaneously in a way that preserves the position-dependent and cell type-specific information at fine scales, thereby greatly improving segmentation accuracy and producing comparable annotations across cell types.

Description: DnpA, a putative de- N -acetylase of the PIG-L superfamily, is required for antibiotic tolerance in Pseudomonas aeruginosa . Exactly how dnpA (gene locus PA5002) directs the formation of antibiotic-tolerant persister cells is currently unknown. Previous research provided evidence for a role in surface-associated process(es), possibly in lipopolysaccharide biosynthesis. In silico sequence analysis of DnpA predicts a single transmembrane domain and N in /C out orientation of DnpA. In contrast, we here show that DnpA is an integral inner membrane protein containing two transmembrane domains, with the major C-terminal part located at the cytoplasmic face. Correct insertion into the inner membrane is necessary for DnpA to promote fluoroquinolone tolerance. The membrane localization of DnpA further supports its role in cell envelope-associated process(es). In addition to shedding light on the biological role of DnpA, this study highlights the risks of overreliance on the predictive value of bioinformatics tools and the importance of rigorous experimental validation of in silico predictions.

Description: Cell wall impermeability and active efflux of drugs are among the primary reasons for drug resistance in mycobacteria. Efflux pumps are tripartite membrane localized transport proteins that expel drug molecules outside the cells. Several of such efflux pumps are annotated in mycobacteria, but few have been characterized, like MSMEG_2991, a putative efflux pump permease of Mycobacterium smegmatis . To substantiate this, we overexpressed MSMEG_2991 protein in Escherichia coli 2443. Expression of MSMEG_2991 elevated the resistance towards structurally unrelated groups of antibiotics. An active antibiotic efflux pump nature of MSMEG_2991 was revealed by assessing the acquisition of ciprofloxacin in the absence and presence of the efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazone, indicating the involvement of proton-motive force (pmf) during the efflux activity. MSMEG_2991 expression elevated biofilm formation in E. coli by 4-fold, keeping parity to some of the earlier reported efflux pumps. In silico analysis suggested the presence of 12 transmembrane helices in MSMEG_2991 resembling EmrD efflux pump of E. coli . Based on in vivo and in silico analyses, MSMEG_2991 may be designated as a pmf-mediated multidrug efflux pump protein that expels diverse groups of antibiotics and might as well be involved in the biofilm enhancement.

Description: Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria–Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I ( pcnB ). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level.

Description: Escherichia coli DedA/Tvp38 family proteins YghB and YqjA are putative membrane transporters with 62% amino acid identity and overlapping functions. An E. coli strain (BC202) with nonpolar yghB and yqjA mutations displays cell-division defects and temperature sensitivity and is sensitive to antibiotics and alkaline pH. In this study, we performed site-directed mutagenesis on conserved, charged amino acids of YqjA and YghB. We discovered two conserved predicted membrane-embedded arginines (R130 and R136) that are critical for function in both proteins as defined by their ability to complement BC202 phenotypes, when expressed from a plasmid. Lysine can substitute for arginine at position R130 indicating a charge dependence at this position, but could not substitute at R136. In light of the established role that arginine plays in the translocation mechanism of numerous membrane transporters, we hypothesize that these amino acids play a role in the transport mechanism of these DedA/Tvp38 family proteins.

Description: Candida albicans is a dimorphic yeast commonly found on human mucosal membranes that switches from yeast to hyphal morphology in response to environmental factors. The change to hyphal growth requires histone H3 modifications by the yeast-specific histone acetyltransferase Rtt109. In addition to its role in morphogenesis, Rtt109-dependent acetylation of histone H3 lysine residues 9 and 56 has regulatory functions during DNA replication and repair. Boric acid (BA) is a broad-spectrum agent that specifically inhibits C. albicans hyphal growth, locking the fungus in its harmless commensal yeast state. The present study characterizes the effect of BA on C. albicans histone acetylation in respect to specificity, time-course and significance. We demonstrate that sublethal concentrations of BA reduce H3K9/H3K56 acetylation, both on a basal level and in response to genotoxic stress. Acetylation at other selected histone sites were not affected by BA. qRT-PCR expression analysis of the DNA repair gene Rad51 indicated no elevated level of genotoxic stress during BA exposure. A forward-mutation analysis demonstrated the BA does not increase spontaneous or induced mutations . The findings suggest that DNA repair remains effective even when histone H3 acetylation decreases and dispels the notion that BA treatment impairs genome integrity in yeast.

Description: The impact of gene mutations on the growth of the cells can be studied using pure cultures. However, the importance of certain proteins and pathways can be also examined via co-culturing wild type and its mutant derivative. Here, the relative fitness of a mutant strain that lacks the global nitrogen regulator, CodY, was examined in Bacillus cereus , a food poisoning Gram-positive bacterium. Fitness measurements revealed that the codY strain was outcompeted when cocultured with the wild-type ATCC 14579 under various rich laboratory medium, and also when inoculated in certain beverages. In nutrient-poor minimal medium, the codY mutant had comparable fitness to the wild-type strain. Interestingly, the relative fitness of the codY strain was antagonistic when it was cultivated in apple or orange juices due to unknown properties of these beverages, highlighting the importance of chemical composition of the test medium during the bacterial fitness measurements.

Description: Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants . On the contrary, transposon insertions in the flagellar structural genes fliP and flgG , that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS , encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB , encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida .

Description: Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens . The studied molecular tools consist of the functional modules luxCDE and luxAB , which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations.

Description: The legume–rhizobial interaction results in the formation of symbiotic nodules in which rhizobia fix nitrogen. During the process of symbiosis, reactive oxygen species (ROS) are generated. Thus, the response of rhizobia to ROS is important for successful nodulation and nitrogen fixation. In this study, we investigated how Azorhizobium caulinodans , a rhizobium that forms both root and stem nodules on its host plant, regulates ROS resistance. We found that in-frame deletions of a gene encoding the putative catalase-peroxidase katG or a gene encoding a LysR-family regulatory protein, oxyR , exhibited increased sensitivity to H 2 O 2 . We then showed that OxyR positively regulated katG expression in an H 2 O 2 -independent fashion. Furthermore, we found that deletion of katG or oxyR led to significant reduction in the number of stem nodules and decrease of nitrogen fixation capacities in symbiosis. Our results revealed that KatG and OxyR are not only critical for antioxidant defense in vitro , but also important for nodule formation and nitrogen fixation during interaction with plant hosts.

Description: Peptidoglycan (also known as murein) is an important envelope component of bacteria, and its turnover usually takes place at considerable levels during normal growth. Amino sugars and murein tripeptide resulting from murein degradation are used for resynthesis of peptidoglycan or as self-generated nutrients or energy sources for cell growth. PgrR (regulator of peptide glycan recycling; formerly YcjZ) was recently identified as a repressor of several genes participating in uptake and degradation of murein tripeptide. In this study, we identified the ycjG gene involved in murein tripeptide degradation as a new direct target of PgrR. The expression of PgrR-regulated genes including ycjY , mppA , mpaA and ycjG was repressed in the presence of a good nitrogen source, but their expression increased under poor nitrogen conditions. Under nitrogen starvation, the pgrR mutant cells exhibited faster growth than wild-type cells, implying that derepression of genes under the control of PgrR may help cells overcome nitrogen limitation. Therefore, these results suggest that nitrogen starvation induces derepression of PgrR-controlled genes involved in uptake and degradation of murein tripeptide, and this may stimulate the utilization of murein tripeptide as a nitrogen source.

Description: A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 x 10 –4 insertions per genome per generation and the rate of IS homologous recombination is 4.5 x 10 –5 recombinations per genome per generation. These events are mostly contributed by the IS elements IS 1 , IS 2 , IS 5 and IS 186 . Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS 186 target-sites, 5'-GGGG(N6/N7)CCCC-3'. We also detected 48 long deletions not involving IS elements.

Description: Extensive and multi-dimensional data sets generated from recent cancer omics profiling projects have presented new challenges and opportunities for unraveling the complexity of cancer genome landscapes. In particular, distinguishing the unique complement of genes that drive tumorigenesis in each patient from a sea of passenger mutations is necessary for translating the full benefit of cancer genome sequencing into the clinic. We address this need by presenting a data integration framework (OncoIMPACT) to nominate patient-specific driver genes based on their phenotypic impact. Extensive in silico and in vitro validation helped establish OncoIMPACT's robustness, improved precision over competing approaches and verifiable patient and cell line specific predictions (2/2 and 6/7 true positives and negatives, respectively). In particular, we computationally predicted and experimentally validated the gene TRIM24 as a putative novel amplified driver in a melanoma patient. Applying OncoIMPACT to more than 1000 tumor samples, we generated patient-specific driver gene lists in five different cancer types to identify modes of synergistic action. We also provide the first demonstration that computationally derived driver mutation signatures can be overall superior to single gene and gene expression based signatures in enabling patient stratification and prognostication. Source code and executables for OncoIMPACT are freely available from http://sourceforge.net/projects/oncoimpact .

Description: Analysis of rewired upstream subnetworks impacting downstream differential gene expression aids the delineation of evolving molecular mechanisms. Cumulative statistics based on conventional differential correlation are limited for subnetwork rewiring analysis since rewiring is not necessarily equivalent to change in correlation coefficients. Here we present a computational method ChiNet to quantify subnetwork rewiring by statistical heterogeneity that enables detection of potential genotype changes causing altered transcription regulation in evolving organisms. Given a differentially expressed downstream gene set, ChiNet backtracks a rewired upstream subnetwork from a super-network including gene interactions known to occur under various molecular contexts. We benchmarked ChiNet for its high accuracy in distinguishing rewired artificial subnetworks, in silico yeast transcription-metabolic subnetworks, and rewired transcription subnetworks for Candida albicans versus Saccharomyces cerevisiae , against two differential-correlation based subnetwork rewiring approaches. Then, using transcriptome data from tolerant S. cerevisiae strain NRRL Y-50049 and a wild-type intolerant strain, ChiNet identified 44 metabolic pathways affected by rewired transcription subnetworks anchored to major adaptively activated transcription factor genes YAP1 , RPN4 , SFP1 and ROX1 , in response to toxic chemical challenges involved in lignocellulose-to-biofuels conversion. These findings support the use of ChiNet in rewiring analysis of subnetworks where differential interaction patterns resulting from divergent nonlinear dynamics abound.

Description: Ribosome biogenesis, a central and essential cellular process, occurs through sequential association and mutual co-folding of protein–RNA constituents in a well-defined assembly pathway. Here, we construct a network of co-evolving nucleotide/amino acid residues within the ribosome and demonstrate that assembly constraints are strong predictors of co-evolutionary patterns. Predictors of co-evolution include a wide spectrum of structural reconstitution events, such as cooperativity phenomenon, protein-induced rRNA reconstitutions, molecular packing of different rRNA domains, protein–rRNA recognition, etc. A correlation between folding rate of small globular proteins and their topological features is known. We have introduced an analogous topological characteristic for co-evolutionary network of ribosome, which allows us to differentiate between rRNA regions subjected to rapid reconstitutions from those hindered by kinetic traps. Furthermore, co-evolutionary patterns provide a biological basis for deleterious mutation sites and further allow prediction of potential antibiotic targeting sites. Understanding assembly pathways of multicomponent macromolecules remains a key challenge in biophysics. Our study provides a ‘proof of concept’ that directly relates co-evolution to biophysical interactions during multicomponent assembly and suggests predictive power to identify candidates for critical functional interactions as well as for assembly-blocking antibiotic target sites.

Description: The emergence of new sequencing technologies has facilitated the use of bacterial whole genome alignments for evolutionary studies and outbreak analyses. These datasets, of increasing size, often include examples of multiple different mechanisms of horizontal sequence transfer resulting in substantial alterations to prokaryotic chromosomes. The impact of these processes demands rapid and flexible approaches able to account for recombination when reconstructing isolates’ recent diversification. Gubbins is an iterative algorithm that uses spatial scanning statistics to identify loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer while concurrently constructing a maximum likelihood phylogeny based on the putative point mutations outside these regions of high sequence diversity. Simulations demonstrate the algorithm generates highly accurate reconstructions under realistically parameterized models of bacterial evolution, and achieves convergence in only a few hours on alignments of hundreds of bacterial genome sequences. Gubbins is appropriate for reconstructing the recent evolutionary history of a variety of haploid genotype alignments, as it makes no assumptions about the underlying mechanism of recombination. The software is freely available for download at github.com/sanger-pathogens/Gubbins , implemented in Python and C and supported on Linux and Mac OS X.

Description: Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.

Description: Genetic variants in or near miRNA genes can have profound effects on miRNA expression and targeting. As user-friendly software for the impact prediction of miRNA variants on a large scale is still lacking, we created a tool called miRVaS. miRVaS automates this prediction by annotating the location of the variant relative to functional regions within the miRNA hairpin (seed, mature, loop, hairpin arm, flanks) and by annotating all predicted structural changes within the miRNA due to the variant. In addition, the tool defines the most important region that is predicted to have structural changes and calculates a conservation score that is indicative of the reliability of the structure prediction. The output is presented in a tab-separated file, which enables fast screening, and in an html file, which allows visual comparison between wild-type and variant structures. All separate images are provided for downstream use. Finally, we tested two different approaches on a small test set of published functionally validated genetic variants for their capacity to predict the impact of variants on miRNA expression.

Description: Analysis of RNA-seq data often detects numerous ‘non-co-linear’ (NCL) transcripts, which comprised sequence segments that are topologically inconsistent with their corresponding DNA sequences in the reference genome. However, detection of NCL transcripts involves two major challenges: removal of false positives arising from alignment artifacts and discrimination between different types of NCL transcripts ( trans -spliced, circular or fusion transcripts). Here, we developed a new NCL-transcript-detecting method (‘NCLscan’), which utilized a stepwise alignment strategy to almost completely eliminate false calls (〉98% precision) without sacrificing true positives, enabling NCLscan outperform 18 other publicly-available tools (including fusion- and circular-RNA-detecting tools) in terms of sensitivity and precision, regardless of the generation strategy of simulated dataset, type of intragenic or intergenic NCL event, read depth of coverage, read length or expression level of NCL transcript. With the high accuracy, NCLscan was applied to distinguishing between trans -spliced, circular and fusion transcripts on the basis of poly(A)- and nonpoly(A)-selected RNA-seq data. We showed that circular RNAs were expressed more ubiquitously, more abundantly and less cell type-specifically than trans -spliced and fusion transcripts. Our study thus describes a robust pipeline for the discovery of NCL transcripts, and sheds light on the fundamental biology of these non-canonical RNA events in human transcriptome.

Description: The central metabolite acetyl phosphate (acP) has long been proposed to influence transcription regulation by directly transferring its phosphoryl group to a number of response regulators in many bacterial species. Here, we provide in vitro evidence for this proposition and demonstrate, using an in vitro transcription system, that acP-dependent phosphorylation of aspartate 51 of CpxR induces transcription of one of its regulon members in E. coli , cpxP . We also used this in vitro transcription system to extend our previously reported in vivo data that hypothesized that acetylation of RNA polymerase (RNAP) influences acP-dependent cpxP transcription, using glutamine as a genetic mimic for acetylated arginine 291 of the carboxy-terminal domain of RNAP α subunit. The data we present here lend strong support to the hypothesis that acP has a direct effect on transcription regulation in E. coli via phosphorylation of CpxR, and that RNAP acetylation can modulate this response.

Description: Chaperones are a diverse class of ubiquitous proteins that assist other cellular proteins in folding correctly and maintaining their native structure. Many different chaperones cooperate to constitute the ‘proteostasis’ machinery in the cells. It has been proposed earlier that archaeal organisms could be ideal model systems for deciphering the basic functioning of the ‘protein folding machinery’ in higher eukaryotes. Several chaperone families have been characterized in archaea over the years but mostly one protein at a time, making it difficult to decipher the composition and mechanistics of the protein folding system as a whole. In order to deal with these lacunae, we have developed a database of all archaeal chaperone proteins, CrAgDb ( C haperone r epertoire in A rchaeal g enomes). The data have been presented in a systematic way with intuitive browse and search facilities for easy retrieval of information. Access to these curated datasets should expedite large-scale analysis of archaeal chaperone networks and significantly advance our understanding of operation and regulation of the protein folding machinery in archaea. Researchers could then translate this knowledge to comprehend the more complex protein folding pathways in eukaryotic systems. The database is freely available at http://14.139.227.92/mkumar/cragdb/ .

Description: In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT ( cysE ) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase ( metA ). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro . Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE .

Description: Detecting allelic biases from high-throughput sequencing data requires an approach that maximises sensitivity while minimizing false positives. Here, we present Allelome.PRO, an automated user-friendly bioinformatics pipeline, which uses high-throughput sequencing data from reciprocal crosses of two genetically distinct mouse strains to detect allele-specific expression and chromatin modifications. Allelome.PRO extends approaches used in previous studies that exclusively analyzed imprinted expression to give a complete picture of the ‘allelome’ by automatically categorising the allelic expression of all genes in a given cell type into imprinted, strain-biased, biallelic or non-informative. Allelome.PRO offers increased sensitivity to analyze lowly expressed transcripts, together with a robust false discovery rate empirically calculated from variation in the sequencing data. We used RNA-seq data from mouse embryonic fibroblasts from F1 reciprocal crosses to determine a biologically relevant allelic ratio cutoff, and define for the first time an entire allelome. Furthermore, we show that Allelome.PRO detects differential enrichment of H3K4me3 over promoters from ChIP-seq data validating the RNA-seq results. This approach can be easily extended to analyze histone marks of active enhancers, or transcription factor binding sites and therefore provides a powerful tool to identify candidate cis regulatory elements genome wide.

Description: Identity by descent (IBD) can be reliably detected for long shared DNA segments, which are found in related individuals. However, many studies contain cohorts of unrelated individuals that share only short IBD segments. New sequencing technologies facilitate identification of short IBD segments through rare variants, which convey more information on IBD than common variants. Current IBD detection methods, however, are not designed to use rare variants for the detection of short IBD segments. Short IBD segments reveal genetic structures at high resolution. Therefore, they can help to improve imputation and phasing, to increase genotyping accuracy for low-coverage sequencing and to increase the power of association studies. Since short IBD segments are further assumed to be old, they can shed light on the evolutionary history of humans. We propose HapFABIA, a computational method that applies biclustering to identify very short IBD segments characterized by rare variants. HapFABIA is designed to detect short IBD segments in genotype data that were obtained from next-generation sequencing, but can also be applied to DNA microarray data. Especially in next-generation sequencing data, HapFABIA exploits rare variants for IBD detection. HapFABIA significantly outperformed competing algorithms at detecting short IBD segments on artificial and simulated data with rare variants. HapFABIA identified 160 588 different short IBD segments characterized by rare variants with a median length of 23 kb (mean 24 kb) in data for chromosome 1 of the 1000 Genomes Project. These short IBD segments contain 752 000 single nucleotide variants (SNVs), which account for 39% of the rare variants and 23.5% of all variants. The vast majority—152 000 IBD segments—are shared by Africans, while only 19 000 and 11 000 are shared by Europeans and Asians, respectively. IBD segments that match the Denisova or the Neandertal genome are found significantly more often in Asians and Europeans but also, in some cases exclusively, in Africans. The lengths of IBD segments and their sharing between continental populations indicate that many short IBD segments from chromosome 1 existed before humans migrated out of Africa. Thus, rare variants that tag these short IBD segments predate human migration from Africa. The software package HapFABIA is available from Bioconductor. All data sets, result files and programs for data simulation, preprocessing and evaluation are supplied at http://www.bioinf.jku.at/research/short-IBD .

Description: Glioma is the most common and fatal primary brain tumour with poor prognosis; however, the functional roles of miRNAs in glioma malignant progression are insufficiently understood. Here, we used an integrated approach to identify miRNA functional targets during glioma malignant progression by combining the paired expression profiles of miRNAs and mRNAs across 160 Chinese glioma patients, and further constructed the functional miRNA–mRNA regulatory network. As a result, most tumour-suppressive miRNAs in glioma progression were newly discovered, whose functions were widely involved in gliomagenesis. Moreover, three miRNA signatures, with different combinations of hub miRNAs (regulations≥30) were constructed, which could independently predict the survival of patients with all gliomas, high-grade glioma and glioblastoma. Our network-based method increased the ability to identify the prognostic biomarkers, when compared with the traditional method and random conditions. Hsa-miR-524-5p and hsa-miR-628-5p, shared by these three signatures, acted as protective factors and their expression decreased gradually during glioma progression. Functional analysis of these miRNA signatures highlighted their critical roles in cell cycle and cell proliferation in glioblastoma malignant progression, especially hsa-miR-524-5p and hsa-miR-628-5p exhibited dominant regulatory activities. Therefore, network-based biomarkers are expected to be more effective and provide deep insights into the molecular mechanism of glioma malignant progression.

Description: A major challenge in cancer genomics is uncovering genes with an active role in tumorigenesis from a potentially large pool of mutated genes across patient samples. Here we focus on the interactions that proteins make with nucleic acids, small molecules, ions and peptides, and show that residues within proteins that are involved in these interactions are more frequently affected by mutations observed in large-scale cancer genomic data than are other residues. We leverage this observation to predict genes that play a functionally important role in cancers by introducing a computational pipeline ( http://canbind.princeton.edu ) for mapping large-scale cancer exome data across patients onto protein structures, and automatically extracting proteins with an enriched number of mutations affecting their nucleic acid, small molecule, ion or peptide binding sites. Using this computational approach, we show that many previously known genes implicated in cancers are enriched in mutations within the binding sites of their encoded proteins. By focusing on functionally relevant portions of proteins—specifically those known to be involved in molecular interactions—our approach is particularly well suited to detect infrequent mutations that may nonetheless be important in cancer, and should aid in expanding our functional understanding of the genomic landscape of cancer.

Description: Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea ( Arabidopsis petraea subsp. umbrosa ), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica . The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated ( r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.

Description: Methods to interpret personal genome sequences are increasingly required. Here, we report a novel framework (EvoTol) to identify disease-causing genes using patient sequence data from within protein coding-regions. EvoTol quantifies a gene's intolerance to mutation using evolutionary conservation of protein sequences and can incorporate tissue-specific gene expression data. We apply this framework to the analysis of whole-exome sequence data in epilepsy and congenital heart disease, and demonstrate EvoTol's ability to identify known disease-causing genes is unmatched by competing methods. Application of EvoTol to the human interactome revealed networks enriched for genes intolerant to protein sequence variation, informing novel polygenic contributions to human disease.

Description: Detecting in vivo transcription factor (TF) binding is important for understanding gene regulatory circuitries. ChIP-seq is a powerful technique to empirically define TF binding in vivo . However, the multitude of distinct TFs makes genome-wide profiling for them all labor-intensive and costly. Algorithms for in silico prediction of TF binding have been developed, based mostly on histone modification or DNase I hypersensitivity data in conjunction with DNA motif and other genomic features. However, technical limitations of these methods prevent them from being applied broadly, especially in clinical settings. We conducted a comprehensive survey involving multiple cell lines, TFs, and methylation types and found that there are intimate relationships between TF binding and methylation level changes around the binding sites. Exploiting the connection between DNA methylation and TF binding, we proposed a novel supervised learning approach to predict TF–DNA interaction using data from base-resolution whole-genome methylation sequencing experiments. We devised beta-binomial models to characterize methylation data around TF binding sites and the background. Along with other static genomic features, we adopted a random forest framework to predict TF–DNA interaction. After conducting comprehensive tests, we saw that the proposed method accurately predicts TF binding and performs favorably versus competing methods.

Description: Enriching target sequences in sequencing libraries via capture hybridization to bait/probes is an efficient means of leveraging the capabilities of next-generation sequencing for obtaining sequence data from target regions of interest. However, homologous sequences from non-target regions may also be enriched by such methods. Here we investigate the fidelity of capture enrichment for complete mitochondrial DNA (mtDNA) genome sequencing by analyzing sequence data for nuclear copies of mtDNA (NUMTs). Using capture-enriched sequencing data from a mitochondria-free cell line and the parental cell line, and from samples previously sequenced from long-range PCR products, we demonstrate that NUMT alleles are indeed present in capture-enriched sequence data, but at low enough levels to not influence calling the authentic mtDNA genome sequence. However, distinguishing NUMT alleles from true low-level mutations (e.g. heteroplasmy) is more challenging. We develop here a computational method to distinguish NUMT alleles from heteroplasmies, using sequence data from artificial mixtures to optimize the method.

Description: Single nucleotide polymorphisms (SNPs) are increasingly used to tag genetic loci associated with phenotypes such as risk of complex diseases. Technically, this is done genome-wide without prior restriction or knowledge of biological feasibility in scans referred to as genome-wide association studies (GWAS). Depending on the linkage disequilibrium (LD) structure at a particular locus, such tagSNPs may be surrogates for many thousands of other SNPs, and it is difficult to distinguish those that may play a functional role in the phenotype from those simply genetically linked. Because a large proportion of tagSNPs have been identified within non-coding regions of the genome, distinguishing functional from non-functional SNPs has been an even greater challenge. A strategy was recently proposed that prioritizes surrogate SNPs based on non-coding chromatin and epigenomic mapping techniques that have become feasible with the advent of massively parallel sequencing. Here, we introduce an R/Bioconductor software package that enables the identification of candidate functional SNPs by integrating information from tagSNP locations, lists of linked SNPs from the 1000 genomes project and locations of chromatin features which may have functional significance. Availability: FunciSNP is available from Bioconductor (bioconductor.org).

Description: Reconstructing the evolutionary relationships of species is a major goal in biology. Despite the increasing number of completely sequenced genomes, a large number of phylogenetic projects rely on targeted sequencing and analysis of a relatively small sample of marker genes. The selection of these phylogenetic markers should ideally be based on accurate predictions of their combined, rather than individual, potential to accurately resolve the phylogeny of interest. Here we present and validate a new phylogenomics strategy to efficiently select a minimal set of stable markers able to reconstruct the underlying species phylogeny. In contrast to previous approaches, our methodology does not only rely on the ability of individual genes to reconstruct a known phylogeny, but it also explores the combined power of sets of concatenated genes to accurately infer phylogenetic relationships of species not previously analyzed. We applied our approach to two broad sets of cyanobacterial and ascomycetous fungal species, and provide two minimal sets of six and four genes, respectively, necessary to fully resolve the target phylogenies. This approach paves the way for the informed selection of phylogenetic markers in the effort of reconstructing the tree of life.

Description: Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

Description: We purified a chitinase from pilei extractions of Coprinopsis cinerea fruiting bodies by ammonium sulfate precipitation and CM Sepharose cation exchange chromatography. MALDI-TOF/TOF MS analysis characterized this purified chitinase as a putative class V chitinase, ChiB1. ChiB1 hydrolyzed colloidal chitin and chitosan, whereas it did not hydrolyze chitin powder. ChiB1 cleaved only pNP-(GlcNAc) 2 , rather than pNP-GlcNAc or pNP-(Glc-NAc) 3 , to release nitrophenol. ChiB1 preferably and progressively released (GlcNAc)2 from (GlcNAc)6 and digested (GlcNAc)6 to two molecules of (GlcNAc)3 in a small proportion, but did not split (GlcNAc) 2 , so it is an exochitinase. ChiB1 has an optimum temperature range of 35°C to 40°C and an optimum pH of 5.0. ChiB1 exhibited Km and Vmax values of 2.63 mg ml –1 and 2.31 μmol min –1  mg protein –1 for colloidal chitin, respectively. The ChiB1 gene, along with another putative endochitinase (class III chitinase gene), was expressed dominantly among eight predicted chitinase genes in the genome, and its expression level increased with the maturation of fruiting bodies. ChiB1 incubation released a large amount of soluble β-glucan fractions from alkali-insoluble cell wall fractions of C. cinerea fruiting bodies, thereby it may promote the degradation of cell walls in synergy with the β-1,3-glucanases during pileus autolysis.

Description: Acidithiobacillus ferrivorans is an acidophile implicated in low-temperature biomining for the recovery of metals from sulfide minerals. Acidithiobacillus ferrivorans obtains its energy from the oxidation of inorganic sulfur compounds, and genes encoding several alternative pathways have been identified. Next-generation sequencing of At. ferrivorans RNA transcripts identified the genes coding for metabolic and electron transport proteins for energy conservation from tetrathionate as electron donor. RNA transcripts suggested that tetrathionate was hydrolyzed by the tetH1 gene product to form thiosulfate, elemental sulfur and sulfate. Despite two of the genes being truncated, RNA transcripts for the SoxXYZAB complex had higher levels than for thiosulfate quinone oxidoreductase ( doxDA genes). However, a lack of heme-binding sites in soxX suggested that DoxDA was responsible for thiosulfate metabolism. Higher RNA transcript counts also suggested that elemental sulfur was metabolized by heterodisulfide reductase ( hdr genes) rather than sulfur oxygenase reductase ( sor ). The sulfite produced as a product of heterodisulfide reductase was suggested to be oxidized by a pathway involving the sat gene product or abiotically react with elemental sulfur to form thiosulfate. Finally, several electron transport complexes were involved in energy conservation. This study has elucidated the previously unknown At. ferrivorans tetrathionate metabolic pathway that is important in biomining.

Description: TRP1 is a frequently used auxotrophic marker for genetic modifications and selections in trp – budding yeast strains, including the commonly used wild-type strain W303a. However, we found that introduction of the TRP1 gene into a trp – strain significantly affected vegetative growth at low and high temperatures. Therefore, caution should be needed when working in a trp – background strain and using the TRP1 marker to study stress response phenotypes, particularly when analyzing temperature sensitivities.

Description: The factors driving the dominance of the Calvin–Benson–Bassham cycle (CBB) or reductive citric acid cycle (rCAC) in autotrophic microorganisms in different habitats are debated. Based on costs for synthesizing a few metabolic intermediates, it has been suggested that the CBB poses a disadvantage due to higher metabolic cost. The purpose of this study was to extend this estimate of cost from metabolite synthesis to biomass synthesis. For 12 gammaproteobacteria (CBB) and five epsilonproteobacteria (rCAC), the amount of ATP to synthesize a gram of biomass from CO 2 was calculated from genome sequences via metabolic maps. The eleven central carbon metabolites needed to synthesize biomass were all less expensive to synthesize via the rCAC (66%–89% of the ATP needed to synthesize them via CBB). Differences in cell compositions did result in differing demands for metabolites among the organisms, but the differences in cost to synthesize biomass were small among organisms that used a particular pathway (e.g. rCAC), compared to the difference between pathways (rCAC versus CBB). The rCAC autotrophs averaged 0.195 moles ATP per g biomass, while their CBB counterparts averaged 0.238. This is the first in silico estimate of the relative expense of both pathways to generate biomass.

Description: Bacteria carry a number of genes that cause cell growth arrest or cell lysis upon expression. Notably, defective prophages retain many lysis proteins. Here, we identified a novel lytic gene, ydfD , on the Qin prophage segment of the Escherichia coli genome. YdfD lyses 99.9% of cells within 2 h of its induction. The co-expression of the upstream gene, dicB , encoding a cell division inhibitor, as well as sulA , encoding another cell division inhibitor, abolished YdfD-induced cell lysis. These results imply that YdfD-induced lysis is a cell division-dependent event. We further found that by deleting the hydrophobic 22-residue N-terminal domain, the resulting 42-residue C-terminal domain was still toxic to cause cell lysis. We propose that YdfD, associated with the cytoplasmic membrane, inhibits an essential cellular process(s).

Description: Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha ). The cell envelope was stained by DiD ® fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation.

Description: Three putative regulatory genes, namely ttmRI, ttmRII and ttmRIII , which are present in the tetramycin ( ttm ) biosynthetic gene cluster, were found in Streptomyces ahygroscopicus . Disruption of ttmRI, ttmRII or ttmRIII reduced tetramycin production, and their complementation restored production to varying degrees. Gene expression analysis of the wild-type (WT) and mutant strains through reverse transcriptase–polymerase chain reaction (RT-PCR) of the ttm gene cluster showed that the expression levels of most of the biosynthetic genes were reduced in ttmRI , ttmRII and ttmRIII . Electrophoretic mobility shift assays demonstrated that TtmRI, TtmRII and TtmRIII bound the promoters of several genes in the ttm gene cluster. This study found that these three proteins are a group of positive regulators that activate the transcription of the ttm gene cluster in S. ahygroscopicus . In addition, ttmRII had a reduced degree of grey pigmentation. Thus, TtmRII has a pleiotropic regulatory function in the tetramycin biosynthetic pathway and in development.

Description: Sulfide:quinone oxidoreductase (SQR) is the primary sulfide-oxidizing enzyme found in all three domains of life. Of the six phylogenetically distinct types of SQR, four have representatives that have been biochemically characterized. The genome of Chlorobaculum tepidum encodes three SQR homologs. One of these, encoded by CT1087, is a type VI SQR that has been previously shown to be required for growth at high sulfide concentrations and to be expressed in sulfide-dependent manner. Therefore, CT1087 was hypothesized to be a high sulfide adapted SQR. CT1087 was expressed in Escherichia coli with an N-terminal His-tag (CT1087NHis 6 ) and purified by Ni-NTA chromatography. CT1087NHis 6 was active and contained FAD as a strongly bound cofactor. The measured kinetic parameters for CT1087NHis 6 indicate a low affinity for sulfide and a high enzymatic turnover rate consistent with the hypothesis for its function inferred from genetic and expression data. These are the first kinetic data for a type VI SQR and have implications for structure-function analyses of all SQR's.

Description: Chloroflexus aggregans is an unbranched multicellular filamentous bacterium having the ability of gliding motility. The filament moves straightforward at a constant rate, ~3 μm sec –1 on solid surface and occasionally reverses the moving direction. In this study, we successfully detected movements of glass beads on the cell-surface along long axis of the filament indicating that the cell-surface movement was the direct force for gliding. Microscopic analyses found that the cell-surface movements were confined to a cell of the filament, and each cell independently moved and reversed the direction. To understand how the cellular movements determine the moving direction of the filament, we proposed a discrete-time stochastic model; sum of the directions of the cellular movements determines the moving direction of the filament only when the filament pauses, and after moving, the filament keeps the same directional movement until all the cells pause and/or move in the opposite direction. Monte Carlo simulation of this model showed that reversal frequency of longer filaments was relatively fixed to be low, but the frequency of shorter filaments varied widely. This simulation result appropriately explained the experimental observations. This study proposed the relevant mechanism adequately describing the motility of the multicellular filament in C. aggregans .

Description: Members of the Gram-positive lactic acid bacteria (LAB) are well-known for their beneficial properties as starter cultures and probiotics. Many LAB species produce ribosomally synthesized proteinaceous antibiotics (bacteriocins). Weissella confusa MBF8-1 is a strain isolated from a fermented soybean product that not only produces useful exopolysaccharides but also exhibits bacteriocin activity, which we call weissellicin MBF. Here, we show that bacteriocin production by W. confusa MBF8-1 is specified by a large plasmid, pWcMBF8-1. Plasmid pWcMBF8-1 (GenBank accession number KR350502), which was identified from the W. confusa MBF8-1 draft genome sequence, is 17 643 bp in length with a G + C content of 34.8% and contains 25 open reading frames (ORFs). Six ORFs constitute the weissellicin MBF locus, encoding three putative double-glycine-motif peptides (Bac1, Bac2, Bac3), an ABC transporter complex (BacTE) and a putative immunity protein (BacI). Two ORFs encode plasmid partitioning and mobilization proteins, suggesting that pWcMBF8-1 is transferable to other hosts. To the best of our knowledge, plasmid pWcMBF8-1 not only represents the first large Weissella plasmid to be sequenced but also the first to be associated with bacteriocin production in W. confusa .

Description: Small non-coding regulatory RNAs (sRNAs) are key players in post-transcriptional regulation of gene expression. Hundreds of sRNAs have been identified in Sinorhizobium meliloti , but their biological function remains unknown for most of them. In this study, we characterized the expression pattern of the gene encoding the 77-nt sRNA MmgR in S. meliloti strain 2011. A chromosomal transcriptional reporter fusion (P mmgR - gfp ) showed that the mmgR promoter is active along different stages of the interaction with alfalfa roots. In pure cultures, P mmgR - gfp activity paralleled the sRNA abundance indicating that mmgR expression is primarily controlled at the level of transcriptional initiation. P mmgR - gfp activity was higher during growth in rhizobial defined medium (RDM) than in TY medium. Furthermore, P mmgR - gfp was induced at 60 min after shifting growing cells from TY to RDM medium, i.e. shorter than the cell doubling time. In defined RDM medium containing NO 3 – , both P mmgR - gfp and MmgR level were repressed by the addition of tryptone or single amino acids, suggesting that mmgR expression depends on the cellular nitrogen (N) status. In silico analysis failed to detect conserved motifs upstream the promoter RNA polymerase binding site, but revealed a strongly conserved motif centered at –28 that may be linked to the observed regulatory pattern by the N source.

Description: A β-glycoside hydrolase was isolated from cell walls material in Coprinopsis cinerea elongating stipes. By analysis of SDS-PAGE, MALDI-TOF/TOF MS and substrate specificity, this enzyme was characterized as an extracellular β-glucosidase which is a trimer consisting of three homosubunits. β-Glucosidase did not degrade β-glucans with modified ends, whereas it hydrolyzed various β-glucans with free ends and related oligosaccharides with β-1,3-, β-1,4- or β-1,6-linkages. Although this β-glucosidase possesses glycosyltransferase activity on laminarioligosaccharides, it did not transfer glucose residues from laminaritriose to β-glucan in stipe cell walls to produce larger β-glucan molecules; instead, it caused a decrease in the molecular size of stipe wall β-glucan by removing glucose. Relatively, the molecular size of wall β-glucans in the elongating apical stipe was less than that found in the non-elongating basal stipes, and this β-glucosidase was more highly expressed in the elongating apical stipe than in non-elongating basal regions. Therefore, we propose that β-glucosidase functions by trimming or cutting the β-glucan side chains on the β-1,3-glucan backbone to prevent them from forming longer branches, keeping the wall plastic to promote diffuse wall growth.

Description: Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans , we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg –1 , while the V max and K m were 7 μM min –1 and 89 ± 7 μM ( n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent ( K i = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents.

Description: Environmental biosurveillance and microbial ecology studies use PCR-based assays to detect and quantify microbial taxa and gene sequences within a complex background of microorganisms. However, the fragmentary nature and growing quantity of DNA-sequence data make group-specific assay design challenging. We solved this problem by developing a software platform that enables PCR-assay design at an unprecedented scale. As a demonstration, we developed quantitative PCR assays for a globally widespread, ecologically important bacterial group in soil, Acidobacteria Group 1. A total of 33 684 Acidobacteria 16S rRNA gene sequences were used for assay design. Following 1 week of computation on a 376-core cluster, 83 assays were obtained. We validated the specificity of the top three assays, collectively predicted to detect 42% of the Acidobacteria Group 1 sequences, by PCR amplification and sequencing of DNA from soil. Based on previous analyses of 16S rRNA gene sequencing, Acidobacteria Group 1 species were expected to decrease in response to elevated atmospheric CO 2 . Quantitative PCR results, using the Acidobacteria Group 1-specific PCR assays, confirmed the expected decrease and provided higher statistical confidence than the 16S rRNA gene-sequencing data. These results demonstrate a powerful capacity to address previously intractable assay design challenges.

Description: Due to dramatic advances in DNA technology, quantitative measures of annotation data can now be obtained in continuous coordinates across the entire genome, allowing various heterogeneous ‘genomic landscapes’ to emerge. Although much effort has been devoted to comparing DNA sequences, not much attention has been given to comparing these large quantities of data comprehensively. In this article, we introduce a method for rapidly detecting local regions that show high correlations between genomic landscapes. We overcame the size problem for genome-wide data by converting the data into series of symbols and then carrying out sequence alignment. We also decomposed the oscillation of the landscape data into different frequency bands before analysis, since the real genomic landscape is a mixture of embedded and confounded biological processes working at different scales in the cell nucleus. To verify the usefulness and generality of our method, we applied our approach to well investigated landscapes from the human genome, including several histone modifications. Furthermore, by applying our method to over 20 genomic landscapes in human and 12 in mouse, we found that DNA replication timing and the density of Alu insertions are highly correlated genome-wide in both species, even though the Alu elements have amplified independently in the two genomes. To our knowledge, this is the first method to align genomic landscapes at multiple scales according to their shape.

Description: The numerous discovered cases of domesticated transposable element (TE) proteins led to the recognition that TEs are a significant source of evolutionary innovation. However, much less is known about the reverse process, whether and to what degree the evolution of TEs is influenced by the genome of their hosts. We addressed this issue by searching for cases of incorporation of host genes into the sequence of TEs and examined the systems-level properties of these genes using the Saccharomyces cerevisiae and Drosophila melanogaster genomes. We identified 51 cases where the evolutionary scenario was the incorporation of a host gene fragment into a TE consensus sequence, and we show that both the yeast and fly homologues of the incorporated protein sequences have central positions in the cellular networks. An analysis of selective pressure (Ka/Ks ratio) detected significant selection in 37% of the cases. Recent research on retrovirus-host interactions shows that virus proteins preferentially target hubs of the host interaction networks enabling them to take over the host cell using only a few proteins. We propose that TEs face a similar evolutionary pressure to evolve proteins with high interacting capacities and take some of the necessary protein domains directly from their hosts.

Description: Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.

Description: The hydrocarbonoclastic bacterium Acinetobacter venetianus RAG-1 has attracted substantial attention due to its powerful oil-degrading capabilities and its potential to play an important ecological role in the cleanup of alkanes. In this study, we compare the transcriptome of the strain RAG-1 grown in dodecane, the corresponding alkanol (dodecanol), and sodium acetate for the characterization of genes involved in dodecane uptake and utilization. Comparison of the transcriptional responses of RAG-1 grown on dodecane led to the identification of 1074 genes that were differentially expressed relative to sodium acetate. Of these, 622 genes were upregulated when grown in dodecane. The highly upregulated genes were involved in alkane catabolism, along with stress response. Our data suggest AlkMb to be primarily involved in dodecane oxidation. Transcriptional response of RAG-1 grown on dodecane relative to dodecanol also led to the identification of permease, outer membrane protein and thin fimbriae coding genes potentially involved in dodecane uptake. This study provides the first model for key genes involved in alkane uptake and metabolism in A. venetianus RAG-1.

Description: Dissimilatory sulfate reduction is the central microbial metabolism in global sulfur cycling. Understanding the importance of sulfate reduction to Earth's biogeochemical S cycle requires aggregating single-cell processes with geochemical signals. For sulfate reduction, these signals include the ratio of stable sulfur isotopes preserved in minerals, as well as the hydrogen isotope ratios and structures of microbial membrane lipids preserved in organic matter. In this study, we cultivated the model sulfate reducer, Desulfovibrio vulgaris DSM 644 T , to investigate how these parameters were perturbed by changes in expression of the protein DsrC. DsrC is critical to the final metabolic step in sulfate reduction to sulfide. S and H isotopic fractionation imposed by the wild type was compared to three mutants. Discrimination against 34 S in sulfate, as calculated from the residual reactant, did not discernibly differ among all strains. However, a closed-system sulfur isotope distillation model, based on accumulated sulfide, produced inconsistent results in one mutant strain IPFG09. Lipids produced by IPFG09 were also slightly enriched in 2 H. These results suggest that DsrC alone does not have a major impact on sulfate-S, though may influence sulfide-S and lipid-H isotopic compositions. While intriguing, a mechanistic explanation requires further study under continuous culture conditions.

Description: Lacticin Q is an unmodified leaderless bacteriocin produced by Lactococcus lactis QU 5. It has been revealed that the production and self-immunity of lacticin Q are facilitated by a gene cluster lnqQBCDEF . The gene for a putative TetR-family transcriptional regulator, termed lnqR , was found nearby the lnqQBCDEF cluster, but its involvement in lacticin Q biosynthesis remained unknown. In this study, we created an LnqR-overexpressing QU 5 recombinant by using lactococcal constitutive promoter P 32 . The recombinant QU 5 showed enhanced production of and self-immunity to lacticin Q. RT-PCR analysis has revealed that an overexpression of LnqR increases the amounts of lnqQBCDEF transcripts, and these six genes are transcribed as an operon in a single transcriptional unit. Interestingly, LnqR expression and thus lacticin Q production by L. lactis QU 5 was found temperature dependent, while LnzR, an LnqR-homologue, in L. lactis QU 14 was expressed in a similar but not identical manner to LnqR, resulting in dissimilar bacteriocin productivities by these strains. This report demonstrates LnqR as the first TetR-family transcriptional regulator involved in LAB bacteriocin biosynthesis and that, as an exceptional case of TetR-family regulators, LnqR positively regulates the transcription of these biosynthetic genes.

Description: Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitrified Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immunoprecipitation protein purification methods. Subsequent chemical and biochemical analyses identified lipid and protein components, respectively. Two major lipid components, comigrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identified as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits; homologous sequences were found only in thermo-acidophilic archaeons. However, a profile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-profile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However, no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet.

Description: Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of Tn phoA -132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE , gidA and truA , (ii) DNA repair; recA and recC , (iii) toxin–antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC . In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase ( cya ) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD , gloA and sodB . The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO.

Description: The culturability of Escherichia coli , Ralstonia eutropha and Bacillus subtilis after incubation in phosphate-buffered saline at either 5°C or 30°C was determined. The culturability of B. subtilis showed little dependence on temperature. The culturability of E. coli rapidly decreased at 30°C but remained almost constant at 5°C. In contrast, the culturability of R. eutropha decreased by three orders of magnitude at 5°C within 24 h but only moderately decreased (one order of magnitude) at 30°C. Remarkably, prolonged incubation of R. eutropha at 30°C resulted in a full recovery of colony forming units in contrast to only a partial recovery at 5°C. Ralstonia eutropha cells at 30°C remained culturable for 3 weeks while culturability at 5°C constantly decreased. The effect of temperature was significantly stronger in a polyhydroxybutyrate-negative mutant. Our data show that accumulated polyhydroxybutyrate has a cold-protective function and can prevent R. eutropha entering the viable but not culturable state.

Description: A number of studies have shown that transcriptome analysis in terms of chromosomal location can reveal regions of non-random transcriptional activity within the genome. Genomic clusters of differentially expressed genes can identify genomic patterns of structural organization, underlying copy number variations or long-range epigenetic regulation such as X-chromosome inactivation. Here we apply an integrative bioinformatics analysis to a collection of 315 freely available mouse pluripotent stem cell samples to discover transcriptional clusters in the genome. We show that over half of the analysed samples (56.83%) carry whole or partial-chromosome spanning clusters which recur in genomic regions previously implicated in chromosomal imbalances. Strikingly, we found that the presence of such large-clusters is linked to the differential expression of a limited number of genes, common to all samples carrying clusters irrespectively of the chromosome where the cluster is found. We have used these genes to train and test classification models that can predict samples that carry large-scale clusters on any chromosome with over 90% accuracy. Our findings suggest that there is a common downstream activation in these cells that affects a limited number of nodes. We propose that this effect is linked to selective advantage and identify potential driver genes.

Description: The rapidly growing amount of genomic sequence data being generated and made publicly available necessitate the development of new data storage and archiving methods. The vast amount of data being shared and manipulated also create new challenges for network resources. Thus, developing advanced data compression techniques is becoming an integral part of data production and analysis. The HapMap project is one of the largest public resources of human single-nucleotide polymorphisms (SNPs), characterizing over 3 million SNPs genotyped in over 1000 individuals. The standard format and biological properties of HapMap data suggest that a dedicated genetic compression method can outperform generic compression tools. We propose a compression methodology for genetic data by introducing H ap Z ipper , a lossless compression tool tailored to compress HapMap data beyond benchmarks defined by generic tools such as gzip , bzip2 and lzma . We demonstrate the usefulness of H ap Z ipper by compressing HapMap 3 populations to 〈5% of their original sizes. H ap Z ipper is freely downloadable from https://bitbucket.org/pchanda/hapzipper/downloads/HapZipper.tar.bz2 .

Description: Spliced alignment plays a central role in the precise identification of eukaryotic gene structures. Even though many spliced alignment programs have been developed, recent rapid progress in DNA sequencing technologies demands further improvements in software tools. Benchmarking algorithms under various conditions is an indispensable task for the development of better software; however, there is a dire lack of appropriate datasets usable for benchmarking spliced alignment programs. In this study, we have constructed two types of datasets: simulated sequence datasets and actual cross-species datasets. The datasets are designed to correspond to various real situations, i.e. divergent eukaryotic species, different types of reference sequences, and the wide divergence between query and target sequences. In addition, we have developed an extended version of our program Spaln , which incorporates two additional features to the scoring scheme of the original version, and examined this extended version, Spaln2, together with the original Spaln and other representative aligners based on our benchmark datasets. Although the effects of the modifications are not individually striking, Spaln2 is consistently most accurate and reasonably fast in most practical cases, especially for plants and fungi and for increasingly divergent pairs of target and query sequences.

Description: Various ‘omics’ technologies, including microarrays and gas chromatography mass spectrometry, can be used to identify hundreds of interesting genes, proteins and metabolites, such as differential genes, proteins and metabolites associated with diseases. Identifying metabolic pathways has become an invaluable aid to understanding the genes and metabolites associated with studying conditions. However, the classical methods used to identify pathways fail to accurately consider joint power of interesting gene/metabolite and the key regions impacted by them within metabolic pathways. In this study, we propose a powerful analytical method referred to as Subpathway-GM for the identification of metabolic subpathways. This provides a more accurate level of pathway analysis by integrating information from genes and metabolites, and their positions and cascade regions within the given pathway. We analyzed two colorectal cancer and one metastatic prostate cancer data sets and demonstrated that Subpathway-GM was able to identify disease-relevant subpathways whose corresponding entire pathways might be ignored using classical entire pathway identification methods. Further analysis indicated that the power of a joint genes/metabolites and subpathway strategy based on their topologies may play a key role in reliably recalling disease-relevant subpathways and finding novel subpathways.

Description: Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (T m ) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique T m when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective T m values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.

Description: The chromatin structure of eukaryotic telomeres plays an essential role in telomere functions. However, their study might be impaired by the presence of interstitial telomeric sequences (ITSs), which have a widespread distribution in different model systems. We have developed a simple approach to study the chromatin structure of Arabidopsis telomeres independently of ITSs by analyzing ChIP-seq data. This approach could be used to study the chromatin structure of telomeres in some other eukaryotes. The analysis of ChIP-seq experiments revealed that Arabidopsis telomeres have higher density of histone H3 than centromeres, which might reflects their short nucleosomal organization. These experiments also revealed that Arabidopsis telomeres have lower levels of heterochromatic marks than centromeres (H3K9 Me2 and H3K27 Me ), higher levels of some euchromatic marks (H3K4 Me2 and H3K9Ac) and similar or lower levels of other euchromatic marks (H3K4 Me3 , H3K36 Me2 , H3K36 Me3 and H3K18Ac). Interestingly, the ChIP-seq experiments also revealed that Arabidopsis telomeres exhibit high levels of H3K27 Me3 , a repressive mark that associates with many euchromatic genes. The epigenetic profile of Arabidopsis telomeres is closely related to the previously defined chromatin state 2. This chromatin state is found in 23% of Arabidopsis genes, many of which are repressed or lowly expressed. At least, in part, this scenario is similar in rice.

Description: Corynebacterium glutamicum is able to metabolize different nitrogen and carbon sources. In standard minimal media, ammonium and urea typically serve as nitrogen source and glucose or sucrose as carbon and energy source; however, amino acids might also play a role as nitrogen, carbon and energy source. In this study, the function of the putative glutaminase GlsK was investigated. A glsK deletion strain showed impaired growth with L-glutamine as carbon and energy source, while growth was improved upon glsK overexpression. GlsK possesses a carboxy-terminal domain that seems to be restricted to Corynebacterium species . A truncated GlsK lacking this extension led to faster growth, indicating a regulatory function of this domain. In fact, GlsK activity is regulated in a pH-dependent manner depending on the carboxy-terminal extension, and is positively influenced by cAMP. Furthermore, the C-terminal extension seems to be important for oligomerization of GlsK.

Description: Sulfur is an essential element for growth and many physiological functions. As sulfur sources for Escherichia coli and related bacteria, specific transporters import various sulfur-containing compounds from the environment. In this study, we identified and characterized an alternative function of the cystine transporter YdjN in E. coli as a transporter of S -sulfocysteine, a sulfur-containing intermediate in the assimilatory cysteine biosynthesis that is used as a sulfur source for the growth of E. coli . We also demonstrated that the transport of S -sulfocysteine via YdjN depends on the transcriptional regulator CysB, a master regulator that controls most of the genes involved in sulfur assimilation and cysteine metabolism. We found that the use of S -sulfocysteine as a sulfur source depends on glutathione because mutations in glutathione biosynthetic genes abolish growth when S -sulfocysteine is used as a sole sulfur source, thereby supporting the previous findings that the conversion of S -sulfocysteine to cysteine is catalyzed by glutaredoxins. To the best of our knowledge, this is the first report of a functional S -sulfocysteine transporter across organisms, which strongly supports the hypothesis that S -sulfocysteine is not only a metabolic intermediate but also a physiologically significant substance in specific natural environments.

Description: Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/ .

Description: Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.

Description: Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff , (M1) and Hivep1, Nfil3, Prdm1 , (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

Description: The RAG1/RAG2 endonuclease initiates V(D)J recombination at antigen receptor loci but also binds to thousands of places outside of these loci. RAG2 localizes directly to lysine 4 trimethylated histone 3 (H3K4me3) through a plant homeodomain (PHD) finger. The relative contribution of RAG2-dependent and RAG1-intrinsic mechanisms in determining RAG1 binding patterns is not known. Through analysis of deep RAG1 ChIP-seq data, we provide a quantitative description of the forces underlying genome-wide targeting of RAG1. Surprisingly, sequence-specific DNA binding contributes minimally to RAG1 targeting outside of antigen receptor loci. Instead, RAG1 binding is driven by two distinct modes of interaction with chromatin: the first is driven by H3K4me3, promoter-focused and dependent on the RAG2 PHD, and the second is defined by H3K27Ac, enhancer-focused and dependent on ‘non-core’ portions of RAG1. Based on this and additional chromatin and genomic features, we formulated a predictive model of RAG1 targeting to the genome. RAG1 binding sites predicted by our model correlate well with observed patterns of RAG1-mediated breaks in human pro-B acute lymphoblastic leukemia. Overall, this study provides an integrative model for RAG1 genome-wide binding and off-target activity and reveals a novel role for the RAG1 non-core region in RAG1 targeting.

Description: Pseudomonas plecoglossicida is a facultative fish pathogen. Recent studies showed that P. plecoglossicida infection in fish was associated with temperature. The aim of this study was to compare the secretomes of P. plecoglossicida cultured in vitro at representative temperatures for pathogenic (20°C) and less pathogenic (30°C) phenotypes. Thirteen proteins in the culture supernatants of P. plecoglossicida showed significant difference in abundance at 20 vs. 30°C. Four proteins were strongly increased at 20°C, including two hemolysin co-regulated proteins (Hcp) that are part of the bacterial type VI secretion system (T6SS), flagellin and an unknown protein. Immunoblot analysis verified the induced secretion of Hcps at 20°C. Furthermore, the upregulation of Hcps at 20°C was confirmed at transcriptional level by RT-qPCR analysis, which also demonstrated the induction of expression of other T6SS-related genes at 20°C. Taken together, we demonstrate the presence of two functionally active T6SS proteins in fish pathogenic P. plecoglossicida strains, as evidenced by the secretion of the T6SS substrate Hcp, the production of which were found to be controlled by temperature. Our findings also support efforts to develop vaccines targeting secreted virulence factors as prophylactic strategies for diseases in fish caused by P. plecoglossicida .

Description: Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using Rel MSM enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to Rel MSM by isothermal titration calorimetry. Enzyme kinetics was followed where K 0.5 was found to be increased with the concomitant reduction of V max value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here.

Description: Although nitric oxide (NO) is an important signaling molecule in bacteria and higher organisms, excessive intracellular NO is highly reactive and dangerous. Therefore, living cells need strict regulation systems for cellular NO homeostasis. Recently, we discovered that Streptomyces coelicolor A3(2) retains the nitrogen oxide cycle (NO 3 – -〉NO 2 – -〉NO-〉NO 3 – ) and nitrite removal system. The nitrogen oxide cycle regulates cellular NO levels, thereby controlling secondary metabolism initiation (red-pigmented antibiotic, RED production) and morphological differentiation. Nitrite induces gene expression in neighboring cells, suggesting another role for this cycle as a producer of transmittable intercellular communication molecules. Here, we demonstrated that ammonium-producing nitrite reductase (NirBD) is involved in regulating NO homeostasis in S. coelicolor A3(2). NirBD was constitutively produced in culture independently of GlnR, a known transcriptional factor. NirBD cleared the accumulated nitrite from the medium. Nir deletion mutants showed increased NO-dependent gene expression at later culture stages, whereas the wild-type M145 showed decreased expression, suggesting that high NO concentration was maintained in the mutant. Moreover, the nir deletion mutant produced more RED than that produced by the wild-type M145. These results suggest that NO 2 – removal by NirBD is important to regulate NO homeostasis and to complete NO signaling in S. coelicolor .