Consensus Promoter Sequence Research Articles

Promoter architecture of Drosophila genes regulated by Myocyte enhancer factor-2.

To gain understanding into the mechanisms of transcriptional activation of muscle genes, we sought to determine if genes targeted by the myogenic transcription factor Myocyte enhancer factor-2 (MEF2) were enriched for specific core promoter elements. We identified 330 known MEF2 target promoters in Drosophila, and analyzed them for for the presence and location of 17 known consensus promoter sequences. As a control, we also searched all Drosophila RNA polymerase II-dependent promoters for the same sequences. We found that promoter motifs were readily detected in the MEF2 target dataset, and that many of them were slightly enriched in frequency compared to the control dataset. A prominent sequence over-represented in the MEF2 target genes was NDM2, that appeared in over 50% of MEF2 target genes and was 2.5-fold over-represented in MEF2 targets compared to background. To test the functional significance of NDM2, we identified two promoters containing a single copy of NDM2 plus an upstream MEF2 site, and tested the activity of these promoters in vivo. Both the sticks and stones and Kahuli fragments showed strong skeletal myoblast-specific expression of a lacZ reporter in embryos. However, the timing and level of reporter expression was unaffected when the NDM2 site in either element was mutated. These studies identify variations in promoter architecture for a set of regulated genes compared to all RNA polymerase II-dependent genes, and underline the potential redundancy in the activities of some core promoter elements.

Transcriptional activation of budding yeast DDI2/3 through chemical modifications of Fzf1.

DDI2 and DDI3 (DDI2/3) are two identical genes in Saccharomyces cerevisiae encoding cyanamide (CY) hydratase. They are not only highly induced by CY, but also by a DNA-damaging agent methyl methanesulfonate (MMS), and the regulatory mechanism is unknown. In this study, we performed a modified genome-wide genetic synthetic array screen and identified Fzf1 as a zinc-finger transcriptional activator required for CY/MMS-induced DDI2/3 expression. Fzf1 binds to a DDI2/3 promoter consensus sequence CS2 in vivo and in vitro, and this interaction was enhanced in response to the CY treatment. Indeed, experimental over production of Fzf1 alone was sufficient to induce DDI2/3 expression; however, CY and MMS treatments did not cause the accumulation or apparent alteration in migration of cellular Fzf1. To test a hypothesis that Fzf1 is activated by covalent modification of CY and MMS, we performed mass spectrometry of CY/MMS-treated Fzf1 and detected a few modified lysine residues. Amino acid substitutions of these residues revealed that Fzf1-K70A completely abolished MMS-induced and reduced CY-induced DDI2/3 expression, indicating that the Fzf1-K70 methylation activates Fzf1. This study collectively reveals a novel regulatory mechanism by which Fzf1 is activated by chemical modifications and in turn induces the expression of its target genes for detoxification.

Subfamily-specific differential contribution of individual monomers and the tether sequence to mouse L1 promoter activity

BackgroundThe internal promoter in L1 5’UTR is critical for autonomous L1 transcription and initiating retrotransposition. Unlike the human genome, which features one contemporarily active subfamily, four subfamilies (A_I, Gf_I and Tf_I/II) have been amplifying in the mouse genome in the last one million years. Moreover, mouse L1 5’UTRs are organized into tandem repeats called monomers, which are separated from ORF1 by a tether domain. In this study, we aim to compare promoter activities across young mouse L1 subfamilies and investigate the contribution of individual monomers and the tether sequence.ResultsWe observed an inverse relationship between subfamily age and the average number of monomers among evolutionarily young mouse L1 subfamilies. The youngest subgroup (A_I and Tf_I/II) on average carry 3–4 monomers in the 5’UTR. Using a single-vector dual-luciferase reporter assay, we compared promoter activities across six L1 subfamilies (A_I/II, Gf_I and Tf_I/II/III) and established their antisense promoter activities in a mouse embryonic fibroblast cell line and a mouse embryonal carcinoma cell line. Using consensus promoter sequences for three subfamilies (A_I, Gf_I and Tf_I), we dissected the differential roles of individual monomers and the tether domain in L1 promoter activity. We validated that, across multiple subfamilies, the second monomer consistently enhances the overall promoter activity. For individual promoter components, monomer 2 is consistently more active than the corresponding monomer 1 and/or the tether for each subfamily. Importantly, we revealed intricate interactions between monomer 2, monomer 1 and tether domains in a subfamily-specific manner. Furthermore, using three-monomer 5’UTRs, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity.ConclusionsThe laboratory mouse is an important mammalian model system for human diseases as well as L1 biology. Our study extends previous findings and represents an important step toward a better understanding of the molecular mechanism controlling mouse L1 transcription as well as L1’s impact on development and disease.

Quaternary Structure and Deoxyribonucleic Acid-Binding Properties of the Heme-Dependent, CO-Sensing Transcriptional Regulator PxRcoM.

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.

Mitochondrial Transcription of Entomopathogenic Fungi Reveals Evolutionary Aspects of Mitogenomes.

Entomopathogenic fungi and more specifically genera Beauveria and Metarhizium have been exploited for the biological control of pests. Genome analyses are important to understand better their mode of action and thus, improve their efficacy against their hosts. Until now, the sequences of their mitochondrial genomes were studied, but not at the level of transcription. Except of yeasts and Neurospora crassa, whose mt gene transcription is well described, in all other Ascomycota, i.e., Pezizomycotina, related information is extremely scarce. In this work, mt transcription and key enzymes of this function were studied. RT-PCR experiments and Northern hybridizations reveal the transcriptional map of the mt genomes of B. bassiana and M. brunneum species. The mt genes are transcribed in six main transcripts and undergo post-transcriptional modifications to create single gene transcripts. Promoters were determined in both mt genomes with a comparative in silico analysis, including all known information from other fungal mt genomes. The promoter consensus sequence is 5′-ATAGTTATTAT-3′ which is in accordance with the definition of the polycistronic transcripts determined with the experiments described above. Moreover, 5′-RACE experiments in the case of premature polycistronic transcript nad1-nad4-atp8-atp6 revealed the 5′ end of the RNA transcript immediately after the in silico determined promoter, as also found in other fungal species. Since several conserved elements were retrieved from these analyses compared to the already known data from yeasts and N. crassa, the phylogenetic analyses of mt RNA polymerase (Rpo41) and its transcriptional factor (Mtf1) were performed in order to define their evolution. As expected, it was found that fungal Rpo41 originate from the respective polymerase of T7/T3 phages, while the ancestor of Mtf1 is of alpha-proteobacterial origin. Therefore, this study presents insights about the fidelity of the mt single-subunit phage-like RNA polymerase during transcription, since the correct identification of mt promoters from Rpo41 requires an ortholog to bacterial sigma factor, i.e., Mtf1. Thus, a previously proposed hypothesis of a phage infected alpha-proteobacterium as the endosymbiotic progenitor of mitochondrion is confirmed in this study and further upgraded by the co-evolution of the bacterial (Mtf1) and viral (Rpo41) originated components in one functional unit.

Case report of recurrent bleeding in an infant with isolated prolonged prothrombin time due to congenital factor VII deficiency---a riddle solved

ABSTRACTFactor VII deficiency is a rare bleeding disorder showing an autosomal recessive pattern of inheritance. Data from our country on this particular entity is lacking. Especially the specific mutations associated with this disease are not well documented. The disease can have a wide spectrum of presentation from asymptomatic to catastrophic central nervous system or gastrointestinal system bleed. It can often present early in the neonatal period or be detected quite later in life. The genotype and phenotype correlation is also not well understood. Here, we report a case of recurrent bleeding in an infant boy who was otherwise absolutely well. His investigations had revealed isolated prolonged prothrombin time which remained uncorrected despite administration of injection vit K. Specific assay for factor VII level revealed that its value less than 1%. Sequencing of the F7 gene revealed our patient to be homozygous for mutation of promoter consensus sequence of F7 gene (-94C > T).

Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12.

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of ‘constitutive’ promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ70), RpoS (σ38), RpoH (σ32), RpoF (σ28) or RpoE (σ24), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ54), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61–81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the ‘repressive’ promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.

Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile.

The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5′-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5′ untranslated regions. By following an in silico iterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors of C. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile, we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.

Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion.

NGFI-A binding protein 2 (NAB2) represses the transcriptional activation of early growth response protein-1 (EGR1), a tumor-suppressor. However, Epidermal Growth Factor (EGF) promotes tumor progression even with significant EGR1 upregulation. The molecular mechanism through which NAB2 is involved in cancer is largely unknown. Therefore, we evaluated how the NAB2-mediated suppression of EGR1 facilitates head and neck squamous cell carcinoma (HNSCC) cancer progression, in association with Sp1, which competes with EGR1 as a transcriptional regulator. The effect of NAB2 on EGR1/SP1 binding to the consensus promoter sequences of MMP2 and MMP9 was evaluated by chromatin immunoprecipitation (ChIP) and promoter luciferase assay. The correlation between EGR1-NAB2 expression and metastatic status was investigated using The Cancer Genome Atlas (TCGA) for HNSCC patients. Our data showed that NAB2 knockdown in FaDu and YD-10B HNSCC cells alleviated EGF-dependent increase of Matrigel invasion. In addition, NAB2 upregulation in EGF-treated FaDu cell diminishes EGR1 transcriptional activity, resulting in the upregulation of Sp1-dependent tumor-promoting genes. TCGA data analysis of 483 HNSCC tumors showed that higher levels of both EGR1 and NAB2 mRNA were significantly associated with metastasis, corresponding to in vitro results. Our data suggest that NAB2 upregulation facilitates EGF-mediated cancer cell invasion through the transactivation of Sp1-dependent tumor-promoting genes. These results provide insight into the paradoxical roles of EGF-EGR1 in cancer progression.

Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032.

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σH in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters.

126 - Selectively targeting the malignant clone through the cellular redox state

Antioxidant compounds that can mimic the activity of endogenous enzymes via redox cycling offer a novel method of targeted cancer treatment. We investigated the therapeutic effect of a mitochondrial- targeted, MnSOD mimetic, Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin, (MnTnBuOE) within myeloid lineage hematopoietic malignancies and utilize the hematopoietic disorder myelodysplastic syndrome (MDS) for the presentation of mechanistic insights to MnTnBuOE efficacy. Consistent with our previous report, MnTnBuOE treatment offers protection of normal HSCs demonstrated by an increase in both CD34+CD38- and CD34+CD38+ stem/progenitor cell viability. However, similar treatment shows significant cytotoxicity mediated by MnTnBuOE induced hydrogen peroxide within acute myeloid leukemia (AML) and MDS models. Transcriptional array analysis indicates that MnTnBuOE treatment initiates activator protein 1 (AP-1) activity. DNA mediated pull down and immunoprecipitation identifies JunB as a transcriptional mediator of MnTnBuOE induced cytotoxicity. Stable knock down of JunB provides protection against MnTnBuOE induced cytotoxicity confirming JunB’s critical role in MnTnBuOE induced hematopoietic malignant cell death. Precipitation with biotin-1,3-cyclopentadione combined with LC-MS/MS analysis of JunB identified Cys285 as the critical cysteine within the DNA binding region of JunB that is susceptible to oxidation by hydrogen peroxide. This, in concert with co-immunoprecipitation of JunB placed Ref-1 as a protein specifically involved in the redox-modification of JunB. Lentiviral mediated knock down of Ref-1 indicates that the interaction between Ref-1 and JunB is critical for MnTnBuOE induced cytotoxicity. The AP-1 ability to bind consensus promoter sequence regardless of methylation status, indicates MnTnBuOE mediated activation of JunB as a potential alternative therapy for aggressive or chemoresistant malignancies in a p53 independent manner. The finding that MnTnBuOE can exert selective cytotoxicity in malignant cells via redox-dependent differential activation of molecular pathways controlling cell fate will significantly aid further development of safe, effective redox-active therapeutics to prevent normal tissue injury and enhance chemotherapeutic efficacy.

Toward a Universal Structural and Energetic Model for Prokaryotic Promoters

With almost no consensus promoter sequence in prokaryotes, recruitment of RNA polymerase (RNAP) to precise transcriptional start sites (TSSs) has remained an unsolved puzzle. Uncovering the underlying mechanism is critical for understanding the principle of gene regulation. We attempted to search the hidden code in ∼16,500 promoters of 12 prokaryotes representing two kingdoms in their structure and energetics. Twenty-eight fundamental parameters of DNA structure including backbone angles, basepair axis, and interbasepair and intrabasepair parameters were used, and information was extracted from x-ray crystallography data. Three parameters (solvation energy, hydrogen-bond energy, and stacking energy) were selected for creating energetics profiles using in-house programs. DNA of promoter regions was found to be inherently designed to undergo a change in every parameter undertaken for the study, in all prokaryotes. The change starts from some distance upstream of TSSs and continues past some distance from TSS, hence giving a signature state to promoter regions. These signature states might be the universal hidden codes recognized by RNAP. This observation was reiterated when randomly selected promoter sequences (with little sequence conservation) were subjected to structure generation; all developed into very similar three-dimensional structures quite distinct from those of conventional B-DNA and coding sequences. Fine structural details at important motifs (viz. −11, −35, and −75 positions relative to TSS) of promoters reveal novel to our knowledge and pointed insights for RNAP interaction at these locations; it could be correlated with how some particular structural changes at the −11 region may allow insertion of RNAP amino acids in interbasepair space as well as facilitate the flipping out of bases from the DNA duplex.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5′ Capping with NAD+

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E.coli total cellular RNA, we establish that sequence determinants for NCIN capping invivo match the NAD+-capping consensus defined invitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping invitro and invivo.

Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its’ 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host’s transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

Extracytoplasmic function sigma factor σD confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum.

Mycolates are α-branched, β-hydroxylated, long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. They form an outer membrane, which functions as a permeability barrier and confers pathogenic mycobacteria to resistance to antibiotics. Although the mycolate biosynthetic pathway has been intensively studied, knowledge of transcriptional regulation of genes involved in this pathway is limited. Here, we report that the extracytoplasmic function sigma factor σD is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD -binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprised acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase and mycolyltransferases; they were involved in mycolate synthesis. Indeed, deletion or overexpression of sigD encoding σD modified the extractable mycolate amount. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD -dependent promoter. Another σD regulon member, l,d-transpeptidase, conferred lysozyme resistance. Thus, σD modifies peptidoglycan cross-linking and enhances mycolate synthesis to provide resistance to environmental stress.

Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

BackgroundThe plant growth promoting rhizobacterium Paenibacillus riograndensis SBR5 is a promising candidate to serve as crop inoculant. Despite its potential in providing environmental and economic benefits, the species P. riograndensis is poorly characterized. Here, we performed for the first time a detailed transcriptome analysis of P. riograndensis SBR5 using RNA-seq technology.ResultsRNA was isolated from P. riograndensis SBR5 cultivated under 15 different growth conditions and combined together in order to analyze an RNA pool representing a large set of expressed genes. The resultant total RNA was used to generate 2 different libraries, one enriched in 5′-ends of the primary transcripts and the other representing the whole transcriptome. Both libraries were sequenced and analyzed to identify the conserved sequences of ribosome biding sites and translation start motifs, and to elucidate operon structures present in the transcriptome of P. riograndensis. Sequence analysis of the library enriched in 5′-ends of the primary transcripts was used to identify 1082 transcription start sites (TSS) belonging to novel transcripts and allowed us to determine a promoter consensus sequence and regulatory sequences in 5′ untranslated regions including riboswitches. A putative thiamine pyrophosphate dependent riboswitch upstream of the thiamine biosynthesis gene thiC was characterized by translational fusion to a fluorescent reporter gene and shown to function in P. riograndensis SBR5.ConclusionsOur RNA-seq analysis provides insight into the P. riograndensis SBR5 transcriptome at the systems level and will be a valuable basis for differential RNA-seq analysis of this bacterium.

Genome-Wide Analysis of the Transcription Start Sites and Promoter Motifs of Phytoplasmas.

Phytoplasmas are obligate intracellular parasitic bacteria that infect both plants and insects. We previously identified the sigma factor RpoD-dependent consensus promoter sequence of phytoplasma. However, the genome-wide landscape of RNA transcripts, including non-coding RNAs (ncRNAs) and RpoD-independent promoter elements, was still unknown. In this study, we performed an improved RNA sequencing analysis for genome-wide identification of the transcription start sites (TSSs) and the consensus promoter sequences. We constructed cDNA libraries using a random adenine/thymine hexamer primer, in addition to a conventional random hexamer primer, for efficient sequencing of 5'-termini of AT-rich phytoplasma RNAs. We identified 231 TSSs, which were classified into four categories: mRNA TSSs, internal sense TSSs, antisense TSSs (asTSSs), and orphan TSSs (oTSSs). The presence of asTSSs and oTSSs indicated the genome-wide transcription of ncRNAs, which might act as regulatory ncRNAs in phytoplasmas. This is the first description of genome-wide phytoplasma ncRNAs. Using a de novo motif discovery program, we identified two consensus motif sequences located upstream of the TSSs. While one was almost identical to the RpoD-dependent consensus promoter sequence, the other was an unidentified novel motif, which might be recognized by another transcription initiation factor. These findings are valuable for understanding the regulatory mechanism of phytoplasma gene expression.

Synthesis of the compatible solute proline by Bacillus subtilis: point mutations rendering the osmotically controlled proHJ promoter hyperactive.

The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in thelocal DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.

An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells.

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.