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Next-generation sequencing (NGS) is becoming a routine approach in most domains of the life sciences. To ensure reproducibility of results, there is a crucial need to improve the automation of NGS data processing and enable forthcoming studies relying on big datasets. Although user-friendly interfaces now exist, there remains a strong need for accessible solutions that allow experimental biologists to analyze and explore their results in an autonomous and flexible way. The protocols here describe a modular system that enable a user to compose and fine-tune workflows based on SnakeChunks, a library of rules for the Snakemake workflow engine. They are illustrated using a study combining ChIP-seq and RNA-seq to identify target genes of the global transcription factor FNR in Escherichia coli, which has the advantage that results can be compared with the most up-to-date collection of existing knowledge about transcriptional regulation in this model organism, extracted from the RegulonDB database. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

With the rapid growth of massively parallel sequencing technologies, still more laboratories are utilising sequenced DNA fragments for genomic analyses. Interpretation of sequencing data is, however, strongly dependent on bioinformatics processing, which is often too demanding for clinicians and researchers without a computational background. Another problem represents the reproducibility of computational analyses across separated computational centres with inconsistent versions of installed libraries and bioinformatics tools. We propose an easily extensible set of computational pipelines, called SnakeLines, for processing sequencing reads; including mapping, assembly, variant calling, viral identification, transcriptomics, and metagenomics analysis. Individual steps of an analysis, along with methods and their parameters can be readily modified in a single configuration file. Provided pipelines are embedded in virtual environments that ensure isolation of required resources from the host operating system, rapid deployment, and reproducibility of analysis across different Unix-based platforms. SnakeLines is a powerful framework for the automation of bioinformatics analyses, with emphasis on a simple set-up, modifications, extensibility, and reproducibility. The framework is already routinely used in various research projects and their applications, especially in the Slovak national surveillance of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The symbiosis with arbuscular mycorrhizal (AM) fungi improves plants' nutrient uptake. During this process, transcription factors have been highlighted to play crucial roles. Members of the GRAS transcription factor gene family have been reported involved in AM symbiosis, but little is known about SCARECROW-LIKE3 (SCL3) genes belonging to this family in Lotus japonicus. In this study, 67 LjGRAS genes were identified from the L. japonicus genome, seven of which were clustered in the SCL3 group. Three of the seven LjGRAS genes expression levels were upregulated by AM fungal inoculation, and our biochemical results showed that the expression of LjGRAS36 was specifically induced by AM colonization. Functional loss of LjGRAS36 in mutant ljgras36 plants exhibited a significantly reduced mycorrhizal colonization rate and arbuscular size. Transcriptome analysis showed a deficiency of LjGRAS36 led to the dysregulation of the gibberellic acid signal pathway associated with AM symbiosis. Together, this study provides important insights for understanding the important potential function of SCL3 genes in regulating AM symbiotic development. [ABSTRACT FROM AUTHOR]

Key message: An AP2 family gene CBX1 is involved in mycorrhizal symbiosis and growth of Lotus japonicus. APETALA 2 (AP2) transcriptional regulator is highly conserved in plants. CBX1 from Lotus japonicus is a member of AP2 family. AMF (Arbuscular mycorrhizal fungi) inoculation experiment demonstrated that expression of CBX1 was significantly induced by AMF. Further promoter analysis showed that the – 764 to − 498 bp region of the CBX1 promoter containing CTTC motif is the AMF responsive region. Functional analysis of cbx1 mutant suggested CBX1 is critical for mycorrhizal symbiosis, especially for arbuscule formation. Moreover, under noncolonized condition, overexpression of CBX1 reduced the root length of L. japonicus but increased the size of root system and shoot length, whereas cbx1 mutant reduced the root size and shoot length, but not effect on root length. In addition, cbx1 altered activity of monolignol biosynthetic gene and increased lignin levels. Collectively, these data indicated that CBX1 is a positive regulator of symbiotic activity and plays roles in the growth of L. japonicus. [ABSTRACT FROM AUTHOR]

Zinc (Zn) deficiency is a widespread agricultural problem in arable soils of the whole world. However, the molecular mechanisms underlying Zn‐deficiency response are largely unknown. Here, we analyzed the transcriptomic profilings of soybean leaves and roots in response to Zn deficiency through Illumina's high‐throughput RNA sequencing in order to understand the molecular basis of Zn‐deficiency response in the plants. A total of 614 and 1011 gene loci were found to be differentially expressed in leaves and roots, respectively, and 88 loci were commonly found in both leaves and roots. Twelve differentially expressed genes (DEGs) were randomly selected for validation by quantitative reverse transcription polymerase chain reaction, and their fold changes were similar to those of RNA‐seq. Gene ontology enrichment analysis showed that ion transport, nicotianamine (NA) biosynthetic process and queuosine biosynthetic process were enriched in the upregulated genes, while oxidation–reduction process and defense response were enriched in the downregulated genes. Among the DEGs, 20 DEGs are potentially involved in Zn homeostasis, including seven ZRT, IRT‐related protein (ZIP) transporter genes, three NA synthase genes, and seven metallothionein genes; 40 DEGs are possibly involved in diverse hormonal signals such as auxin, cytokinin, ethylene and gibberellin; nine DEGs are putatively involved in calcium signaling; 85 DEGs are putative transcription factor genes. Nine DEGs were found to contain zinc‐deficiency‐response element in their promoter regions. These results could provide comprehensive insights into the soybean response to Zn deficiency and will be helpful for further elucidation of the molecular mechanisms of Zn‐deficiency response and Zn‐deficiency tolerance in plants. [ABSTRACT FROM AUTHOR]

Background: In living organisms, small heat shock proteins (sHSPs) are triggered in response to stress situations. This family of proteins is large in plants and, in the case of tomato (Solanum lycopersicum), 33 genes have been identified, most of them related to heat stress response and to the ripening process. Transcriptomic and proteomic studies have revealed complex patterns of expression for these genes. In this work, we investigate the coregulation of these genes by performing a computational analysis of their promoter architecture to find regulatory motifs known as heat shock elements (HSEs). We leverage the presence of sHSP members that originated from tandem duplication events and analyze the promoter architecture diversity of the whole sHSP family, focusing on the identification of HSEs. Results: We performed a search for conserved genomic sequences in the promoter regions of the sHSPs of tomato, plus several other proteins (mainly HSPs) that are functionally related to heat stress situations or to ripening. Several computational analyses were performed to build multiple sequence motifs and identify transcription factor binding sites (TFBS) homologous to HSF1AE and HSF21 in Arabidopsis. We also investigated the expression and interaction of these proteins under two heat stress situations in whole tomato plants and in protoplast cells, both in the presence and in the absence of heat shock transcription factor A2 (HsfA2). The results of these analyses indicate that different sHSPs are up-regulated depending on the activation or repression of HsfA2, a key regulator of HSPs. Further, the analysis of protein-protein interaction between the sHSP protein family and other heat shock response proteins (Hsp70, Hsp90 and MBF1c) suggests that several sHSPs are mediating alternative stress response through a regulatory subnetwork that is not dependent on HsfA2. Conclusions: Overall, this study identifies two regulatory motifs (HSF1AE and HSF21) associated with the sHSP family in tomato which are considered genomic HSEs. The study also suggests that, despite the apparent redundancy of these proteins, which has been linked to gene duplication, tomato sHSPs showed different up-regulation and different interaction patterns when analyzed under different stress situations. [ABSTRACT FROM AUTHOR]

Abstract: DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double‐strand breaks. Similar such helicase activity for resolving secondary structures and structure‐specific nuclease activity are needed during DNA replication to process the chromosome‐specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA. The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2‐mediated clean‐up mechanisms impairs centromeric DNA replication and CENP‐A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late‐S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Tacrolimus (FK506), an effective immunosuppressant, is widely used in the treatment of autoimmune diseases. In this study, we identified that BulZ, a Streptomyces antibiotic regulatory protein (SARP) family regulator, acted as a positive regulator for spore differentiation and tacrolimus production. A knockout of bulZ resulted in a 47.5% decrease of tacrolimus production and a delay of spore differentiation. Using quantitative real-time PCR (qRT-PCR) analysis and electrophoretic mobility shift assays (EMSAs), it was found that BulZ directly activated the transcriptions of bulZ and bulS2, a putative γ-butyrolactone (GBL) synthetase, and bulS2 was shown to play a positive role in tacrolimus biosynthesis. Meanwhile, BulZ was able to indirectly regulate the transcriptions of the cluster-linked activator genes tcs7 and fkbN, as well as the GBL receptor gene bulR1. STSU_RS22595, which encoded a WhiB family transcriptional regulator, was found to be a previously unknown potential target gene of BulZ based on a whole-genome search of the conserved sequence (5′-TSVAVVVNVNBTSRAGNN-3′) of the SARP-binding motifs. Although overexpression of STSU_RS22595 did not result in an obvious enhancement of tacrolimus yield, STSU_RS22595 might have important effects on the spore differentiation process. Finally, co-overexpression of bulZ and its target gene bulS2 improved tacrolimus production by 36% compared to the control strain, reaching 324 mg/L. The insights obtained in this study will help further elucidate the regulatory mechanism of tacrolimus biosynthesis and provide new avenues for further improvement of tacrolimus production. [ABSTRACT FROM AUTHOR]

Diatoms are amongst the most important marine microalgae in terms of biomass, but little is known concerning the molecular mechanisms that regulate their versatile metabolism. Here, the pennate diatom Phaeodactylum tricornutum was studied at the metabolite and transcriptome level during nitrogen starvation and following imposition of three other stresses that impede growth. The coordinated upregulation of the tricarboxylic acid (TCA) cycle during the nitrogen stress response was the most striking observation. Through co-expression analysis and DNA binding assays, the transcription factor bZIP14 was identified as a regulator of the TCA cycle, also beyond the nitrogen starvation response, namely in diurnal regulation. Accordingly, metabolic and transcriptional shifts were observed upon overexpression of bZIP14 in transformed P. tricornutum cells. Our data indicate that the TCA cycle is a tightly regulated and important hub for carbon reallocation in the diatom cell during nutrient starvation and that bZIP14 is a conserved regulator of this cycle. [ABSTRACT FROM AUTHOR]

Tacrolimus (FK506) is a 23-membered macrolide immunosuppressant used in current clinics. Understanding how the tacrolimus biosynthetic gene cluster is regulated is important to increase its industrial production. Here, we analysed the effect of the disruption of fkbN (encoding a LAL-type positive transcriptional regulator) on the whole transcriptome of the tacrolimus producer Streptomyces tsukubaensis using microarray technology. Transcription of fkbN in the wild type strain increases from 70 h of cultivation reaching a maximum at 89 h, prior to the onset of tacrolimus biosynthesis. Disruption of fkbN in S. tsukubaensis does not affect growth but prevents tacrolimus biosynthesis. Inactivation of fkbN reduces the transcription of most of the fkb cluster genes, including some all (for allylmalonyl-CoA biosynthesis) genes but does not affect expression of allMNPOS or fkbR (encoding a LysR-type regulator). Disruption of fkbN does not suppress transcription of the cistron tcs6-fkbQ-fkbN; thus, FkbN self-regulates only weakly its own expression. Interestingly, inactivation of FkbN downregulates the transcription of a 4′-phosphopantetheinyl transferase coding gene, which product is involved in tacrolimus biosynthesis, and upregulates the transcription of a gene cluster containing a cpkA orthologous gene, which encodes a PKS involved in coelimycin P1 biosynthesis in Streptomyces coelicolor. We propose an information theory-based model for FkbN binding sequences. The consensus FkbN binding sequence consists of 14 nucleotides with dyad symmetry containing two conserved inverted repeats of 7 nt each. This FkbN target sequence is present in the promoters of FkbN-regulated genes. [ABSTRACT FROM AUTHOR]

Background: NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided. Methods: Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points. Results: We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress. Conclusions: The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle. [ABSTRACT FROM AUTHOR]

The regulation of floral organ identity was investigated using a forward genetic approach in five floral homeotic mutants of Thalictrum, a noncore eudicot. We hypothesized that these mutants carry defects in the floral patterning genes. Mutant characterization comprised comparative floral morphology and organ identity gene expression at early and late developmental stages, followed by sequence analysis of coding and intronic regions to identify transcription factor binding sites and protein-protein interaction (PPI) motifs. Mutants exhibited altered expression of floral MADS-box genes, which further informed the function of paralogs arising from gene duplications not found in reference model systems. The ensuing modified BCE models for the mutants supported instances of neofunctionalization (e.g., B-class genes expressed ectopically in sepals), partial redundancy (E-class), or subfunctionalization (C-class) of paralogs. A lack of deleterious mutations in the coding regions of candidate floral MADS-box genes suggested that cis-regulatory or trans-acting mutations are at play. Consistent with this hypothesis, double-flower mutants had transposon insertions or showed signs of transposon activity in the regulatory intron of AGAMOUS (AG) orthologs. Single amino acid substitutions were also found, yet they did not fall on any of the identified DNA binding or PPI motifs. In conclusion, we present evidence suggesting that transposon activity and regulatory mutations in floral homeotic genes likely underlie the striking phenotypes of these Thalictrum floral homeotic mutants., (© 2020 Wiley Periodicals LLC.)

Background: Genome sequencing revealed that Streptomyces sp. can dedicate up to ~ 10% of their genomes for the biosynthesis of bioactive secondary metabolites. However, the majority of these biosynthetic gene clusters are only weakly expressed or not at all. Indeed, the biosynthesis of natural products is highly regulated through integrating multiple nutritional and environmental signals perceived by pleiotropic and pathway-specific transcriptional regulators. Although pathway-specific refactoring has been a proved, productive approach for the activation of individual gene clusters, the construction of a global super host strain by targeting pleiotropic-specific genes for the expression of multiple diverse gene clusters is an attractive approach. Results: Streptomyces albus J1074 is a gifted heterologous host. To further improve its secondary metabolite expression capability, we rationally engineered the host by targeting genes affecting NADPH availability, precursor flux, cell growth and biosynthetic gene transcriptional activation. These studies led to the activation of the native paulomycin pathway in engineered S. albus strains and importantly the upregulated expression of the heterologous actinorhodin gene cluster. Conclusions: Rational engineering of Streptomyces albus J1074 yielded a series of mutants with improved capabilities for native and heterologous expression of secondary metabolite gene clusters. [ABSTRACT FROM AUTHOR]