Juan Miranda-Ríos

RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation

RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database offering curated knowledge of the transcriptional regulatory network of Escherichia coli K12, currently the best-known electronically encoded database of the genetic regulatory network of any free-living organism. This paper summarizes the improvements, new biology and new features available in version 6.0. Curation of original literature is, from now on, up to date for every new release. All the objects are supported by their corresponding evidences, now classified as strong or weak. Transcription factors are classified by origin of their effectors and by gene ontology class. We have now computational predictions for σ54 and five different promoter types of the σ70 family, as well as their corresponding −10 and −35 boxes. In addition to those curated from the literature, we added about 300 experimentally mapped promoters coming from our own high-throughput mapping efforts. RegulonDB v.6.0 now expands beyond transcription initiation, including RNA regulatory elements, specifically riboswitches, attenuators and small RNAs, with their known associated targets. The data can be accessed through overviews of correlations about gene regulation. RegulonDB associated original literature, together with more than 4000 curation notes, can now be searched with the Textpresso text mining engine.

RegulonDB version 7.0: transcriptional regulation of Escherichia coli K-12 integrated within genetic sensory response units (Gensor Units)

RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database of the best-known regulatory network of any free-living organism, that of Escherichia coli K-12. The major conceptual change since 3 years ago is an expanded biological context so that transcriptional regulation is now part of a unit that initiates with the signal and continues with the signal transduction to the core of regulation, modifying expression of the affected target genes responsible for the response. We call these genetic sensory response units, or Gensor Units. We have initiated their high-level curation, with graphic maps and superreactions with links to other databases. Additional connectivity uses expandable submaps. RegulonDB has summaries for every transcription factor (TF) and TF-binding sites with internal symmetry. Several DNA-binding motifs and their sizes have been redefined and relocated. In addition to data from the literature, we have incorporated our own information on transcription start sites (TSSs) and transcriptional units (TUs), obtained by using high-throughput whole-genome sequencing technologies. A new portable drawing tool for genomic features is also now available, as well as new ways to download the data, including web services, files for several relational database manager systems and text files including BioPAX format.

A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria.

The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5′ regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helixes, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.

Detection of 5′- and 3′-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of ∼60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30–65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5′- and 3′-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3′-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3′-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5′ or 3′ ends of the mRNAs with perfect complementarity.

Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or β16–β22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III β16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through β-16 of domain III promoting membrane insertion and cell death.

Small Stress Response Proteins in Escherichia coli: Proteins Missed by Classical Proteomic Studies

Proteins of 50 or fewer amino acids are poorly characterized in all organisms. The corresponding genes are challenging to reliably annotate, and it is difficult to purify and characterize the small protein products. Due to these technical limitations, little is known about the abundance of small proteins, not to mention their biological functions. To begin to characterize these small proteins in Escherichia coli, we assayed their accumulation under a variety of growth conditions and after exposure to stress. We found that many small proteins accumulate under specific growth conditions or are stress induced. For some genes, the observed changes in protein levels were consistent with known transcriptional regulation, such as ArcA activation of the operons encoding yccB and ybgT. However, we also identified novel regulation, such as Zur repression of ykgMO, cyclic AMP response protein (CRP) repression of azuC, and CRP activation of ykgR. The levels of 11 small proteins increase after heat shock, and induction of at least 1 of these, YobF, occurs at a posttranscriptional level. These results show that small proteins are an overlooked subset of stress response proteins in E. coli and provide information that will be valuable for determining the functions of these proteins.

Hydropathic Complementarity Determines Interaction of Epitope 869HITDTNNK876 in Manduca sexta Bt-R1 Receptor with Loop 2 of Domain II ofBacillus thuringiensis Cry1A Toxins

In susceptible insects, Cry toxin specificity correlates with receptor recognition. In previous work, we characterized an scFv antibody (scFv73) that inhibits binding of Cry1A toxins to cadherin-like receptor. The CDR3 region of scFv73 shared homology with an 8-amino acid epitope (869HITDTNNK876) of the Manduca sexta cadherin-like receptor Bt-R1 (Gomez, I., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2001) J. Biol. Chem. 276, 28906–28912). In this work, we show that the previous sequence of scFv73 CDR3 region was obtained from the noncoding DNA strand. However, most importantly, both scFv73 CDR3 amino acid sequences of the coding and noncoding DNA strands have similar binding capabilities to Cry1Ab toxin as Bt-R1 869HITDTNNK876 epitope, as demonstrated by the competition of scFv73 with binding to Cry1Ab with synthetic peptides with amino acid sequences corresponding to these regions. Using synthetic peptides corresponding to three exposed loop regions of domain II of Cry1Aa and Cry1Ab toxins, we found that loop 2 synthetic peptide competed with binding of scFv73 to Cry1A toxins in Western blot experiments. Also, loop 2 mutations that affect toxicity of Cry1Ab toxin are affected in scFv73 binding. Toxin overlay assays of Cry1A toxins to M. sexta brush border membrane proteins showed that loop 2 synthetic peptides competed with binding of Cry1A toxins to cadherin-like Bt-R1 receptor. These experiments identified loop 2 in domain II of as the cognate binding partner of Bt-R1 869HITDTNNK876. Finally, 10 amino acids from β-6-loop 2 region of Cry1Ab toxin (363SSTLYRRPFNI373) showed hydropathic pattern complementarity to a 10-amino acid region of Bt-R1(865NITIHITDTNN875), suggesting that binding of Cry1A toxins to Bt-R1 is determined by hydropathic complementarity and that the binding epitope of Bt-R1 may be larger than the one identified by amino acid sequence similarity to scFv73.

The emerging role of MicroRNAs in the regulation of gene expression by nutrients.

MicroRNAs (miRNAs) are a class of evolutionarily conserved, small non-coding RNAs of 19-24 nucleotides in length that regulate gene expression mostly at the posttranscriptional level. They are known to be involved in the control of different processes such as cell cycling, programmed cell death, cell differentiation, tumor development, metastasis, and sensing of nutrient stress. This review summarizes the evidence regarding the changes in miRNA expression that are caused by diets with a deficiency or augmented intake of nutrients such as amino acids, carbohydrates, fatty acids, vitamins, and phytochemicals. As diet is known to influence the expression of miRNAs, miRNA profiling has the potential to be useful in the assessment of nutritional status in dietary intervention studies. Additionally, as it can change miRNA expression, diet may be used as a therapeutic agent to treat many different diseases. Also, we explored here some ideas on therapeutics based on the manipulation of miRNA expression levels for dietary-derived diseases as well as the putative effect of food-derived miRNAs on host gene expression.

A Bacillus thuringiensis S-layer protein involved in toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

ABSTRACT The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.

Regulation of gene expression in response to oxygen in Rhizobium etli: role of FnrN in fixNOQP expression and in symbiotic nitrogen fixation.

Previously, we reported finding duplicated fixNOQP operons in Rhizobium etli CFN42. One of these duplicated operons is located in the symbiotic plasmid (fixNOQPd), while the other is located in a cryptic plasmid (fixNOQPf). Although a novel FixL-FixKf regulatory cascade participates in microaerobic expression of both fixNOQP duplicated operons, we found that a mutation in fixL eliminates fixNOQPf expression but has only a moderate effect on expression of fixNOQPd. This suggests that there are differential regulatory controls. Interestingly, only the fixNOQPd operon was essential for symbiotic nitrogen fixation (L. Girard, S. Brom, A. Dávalos, O. Lopez, M. Soberón, and D. Romero, Mol. Plant-Microbe Interact. 13:1283-1292, 2000). Searching for potential candidates responsible for the differential expression, we characterized two fnrN homologs (encoding transcriptional activators of the cyclic AMP receptor protein [CRP]-Fnr family) in R. etli CFN42. One of these genes (fnrNd) is located on the symbiotic plasmid, while the other (fnrNchr) is located on the chromosome. Analysis of the expression of the fnrN genes using transcriptional fusions with lacZ showed that the two fnrN genes are differentially regulated, since only fnrNd is expressed in microaerobic cultures of the wild-type strain while fnrNchr is negatively controlled by FixL. Mutagenesis of the two fnrN genes showed that both genes participate, in conjunction with FixL-FixKf, in the microaerobic induction of the fixNOQPd operon. Participation of these genes is also seen during the symbiotic process, in which mutations in fnrNd and fnrNchr, either singly or in combination, lead to reductions in nitrogen fixation. Therefore, R. etli employs a regulatory circuit for induction of the fixNOQPd operon that involves at least three transcriptional regulators of the CRP-Fnr family. This regulatory circuit may be important for ensuring optimal production of the cbb(3), terminal oxidase during symbiosis.