Rafael P. Mellado

Estimation of bacterial diversity using next generation sequencing of 16S rDNA: a comparison of different workflows.

BackgroundNext generation sequencing (NGS) enables a more comprehensive analysis of bacterial diversity from complex environmental samples. NGS data can be analysed using a variety of workflows. We test several simple and complex workflows, including frequently used as well as recently published tools, and report on their respective accuracy and efficiency under various conditions covering different sequence lengths, number of sequences and real world experimental data from rhizobacterial populations of glyphosate-tolerant maize treated or untreated with two different herbicides representative of differential diversity studies.ResultsAlignment and distance calculations affect OTU estimations, and multiple sequence alignment exerts a major impact on the computational time needed. Generally speaking, most of the analyses produced consistent results that may be used to assess differential diversity changes, however, dataset characteristics dictate which workflow should be preferred in each case.ConclusionsWhen estimating bacterial diversity, ESPRIT as well as the web-based workflow, RDP pyrosequencing pipeline, produced good results in all circumstances, however, its computational requirements can make method-combination workflows more attractive, depending on sequence variability, number and length.

Effect of the herbicide glyphosate on glyphosate-tolerant maize rhizobacterial communities: a comparison with pre-emergency applied herbicide consisting of a combination of acetochlor and terbuthylazine.

A comparison was made of the effect of glyphosate (RoundupPlus), a post-emergency applied herbicide, and of HarnessGTZ, a pre-emergency applied herbicide, on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. The potential effect was monitored by direct amplification, cloning and sequencing of soil DNA encoding 16S rRNA, rhizobacterial DNA hybridization to commercially available genome-wide microarrays from the soil bacterium Streptomyces coelicolor, and high-throughput DNA pyrosequencing of the bacterial DNA coding for 16S rRNA hypervariable V6 region. The results obtained strongly suggest that both herbicides do in fact affect the maize rhizobacterial communities, glyphosate being, to a great extent, the environmentally less aggressive herbicide.

Four genes encoding different type I signal peptidases are organized in a cluster in Streptomyces lividans TK21.

Four adjacent genes (sipW, sipX, sipY and sipZ) encoding different type I signal peptidases, were isolated on a 7860 bp DNA fragment from Streptomyces lividans TK21. Three of the sip genes constitute an operon and the fourth is the first gene of another operon encompassing three additional, unrelated genes. A DNA fragment containing the four sip genes complemented an Escherichia coli type I signal peptidase mutant when cloned in a multicopy plasmid. Clustering of four different type I signal peptidase genes seems, so far, to be a unique feature of Streptomyces.

Effect of glucose on agarase overproduction by Streptomyces

The Streptomyces coelicolor dagA gene, coding for an extracellular agarase, has been propagated on a multicopy plasmid in S. coelicolor A3(2), the natural agarase producer strain and in S. lividans TK21, a closely related, nonproducer strain. The effect of the carbon source on the production of agarase by both strains, upon cultivation in liquid medium, revealed that the glucose repression affected the synthesis of agarase at the level of secretion, rather than at the level of transcription. In the presence of glucose, the pre-agarase was degraded intracellularly and the overall secretion of proteases decreased considerably in both strains, suggesting a negative regulatory role for glucose in the overall secretion in Streptomyces.

SipY Is the Streptomyces lividans Type I Signal Peptidase Exerting a Major Effect on Protein Secretion

Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins. The developmental complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (sipW, sipX, sipY, and sipZ) unusually clustered in its chromosome. Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual sip mutants. None of the sip genes seemed to be essential for bacterial growth. Analysis of total extracellular proteins indicated that SipY is likely to be the major S. lividans SPase, since the sipY mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.

Effect of Cry1Ab Protein on Rhizobacterial Communities of Bt-Maize over a Four-Year Cultivation Period

Background Bt-maize is a transgenic variety of maize expressing the Cry toxin from Bacillus turingiensis. The potential accumulation of the relative effect of the transgenic modification and the cry toxin on the rhizobacterial communities of Bt-maize has been monitored over a period of four years. Methodology/Principal Findings The accumulative effects of the cultivation of this transgenic plant have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The obtained sequences were subjected to taxonomic, phylogenetic and taxonomic-independent diversity studies. The results obtained were consistent, indicating that variations detected in the rhizobacterial community structure were possibly due to climatic factors rather than to the presence of the Bt-gene. No variations were observed in the diversity estimates between non-Bt and Bt-maize. Conclusions/Significance The cultivation of Bt-maize during the four-year period did not change the maize rhizobacterial communities when compared to those of the non-Bt maize. This is the first study to be conducted with Bt-maize during such a long cultivation period and the first evaluation of rhizobacterial communities to be performed in this transgenic plant using Next Generation Sequencing.

In vivo transcription of bacteriophage phi 29 DNA: transcription termination.

This work was supported by Community of Madrid (Grupo Estrategico 2000-2003), NIH, grant R01CA77575, and SAF 2001-2245.The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.Trabajo presentado en 44th Annual Meeting Society for Neuroscience, celebrado en Washington, DC (USA) del 15 al 19 de noviembre de 2014Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.Supported by Grant GM-08041 from the National Institutes of Health, United States Public Health Service.The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.Aided by grants from the National Institutes of Health U.S. Public Health Service, and E. I. Du Pont de Neumours and Company, Inc.This work was supported in part by NRSA, National Institutes of Health Grants NS09463 and NS32501 and from National Science Foundation Grant 9310965.We have recently developed a new method to detect and characterize single base substitutions in transcribed genes which is based on the ability of RNAse A to recognize and cleave single base mismatches in RNA:RNA heteroduplexes. The RNAse A misrnatch cleavage assay was applied to screen human colon carcinoma cell lines and primary tumors for the presence of mutant e-X-ras oncogenes. We have determined that the mutant e-X-ras allele is overexpressed and amplified relative to the normal in the SX-CO-l human colon carcinoma cell lineo The oncogene mutation has been characterized by this method as a glycine to valine substitution at codon 12 of the e-X-ras gene. This result was confirmed by cloning and sequencing. We have previously reported that about 40% of primary human colon tumors contain e-X-ras genes mutant at codon 12 (Forrester et al, Nature 327: 298, 1987). We report here the characterization by molecular cloning and sequencing of the mutation in the e-X-ras oneogene from two of these tumors (tumors 3 and 28). We also describe the histopathologieal eharaeterization of these two tumors and demonstrate, by Southern blot hybridization of NIH3T3 transformants, the simultaneous presenee of mutant e-X-ras and N-ras oncogenes in villous adenoma 28. Our results provide evidence for the frequent assoeiation of ras somatie mutational aetivation in the early stages of tumor development in this common type of human eaneer.Aided by Grants AM-01845, AM-08953, and l-Sol-FR-05099 from the National Institutes o f Health, United States Public Health Service, and E. I. Du Pont de Nemours and Company, Inc. A preliminary report o f this work was presented at the Second Meeting o f the Federation o f European Biochemical Societies (symposium on “Ribonucleic Acid-Structure and Function”), Vienna, April 21 to 24, 1965.1 pagina.-- Trabajo presentado al: 4th International Meeting on Apicomplexa in Farm Animals. (Madrid, Spain. 11-14 October ,2017).Supported by Grant GM-08041 from the National Institutes of Health, United States Public Health Service.Resumen del trabajo presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.Poster presentado al Annual Biomedical Research Conference for Minority Students celebrado en California (US) del 7 al 10 de noviembre de 2012.The phage phi 29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP alpha subunit (alpha-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the alpha-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a -35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression.Resumen del poster presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production and retard aging in a variety of species. It has been proposed that alterations in membrane saturation are central to these actions of CR. As a step towards testing this theory, mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN-93G diets at 95 % (control) or 60 % (CR) of ad libitum for 8 months. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. Skeletal muscle mitochondrial lipid composition, proton leak, and H(2)O(2) production were measured. Phospholipid fatty acid composition in CR mice was altered in a manner that reflected the n-3 and n-6 fatty acid profiles of their respective dietary lipid sources. Dietary lipid composition did not alter proton leak kinetics between the CR groups. However, the capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to the other CR groups. The results of this study indicate that dietary lipid composition can influence ROS production in muscle mitochondria of CR mice. It remains to be determined if lard or other dietary oils can maximize the CR-induced decreases in ROS production.To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozymez substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min 21 ) were typically 15-fold greater than cleavage rates (0.2 min 21 ), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (k on 5 4 3 10 8 M 21 min 21 ), and the ribozymez substrate complex is very stable (K D < 25 pM ;k off < 0.01 min 21 ). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.Lazarillo, a protein recognized by the monoclonal antibody 10E6, is expressed by a subset of neurons in the developing nervous system of the grasshopper. It is a glycoprotein of 45x10(3) M(r) with internal disulfide bonds and linked to the extracellular side of the plasma membrane by a glycosylphosphatidylinositol moiety. Peptide sequences obtained from affinity purified adult protein were used to identify an embryonic cDNA clone, and in situ hybridizations confirmed that the distribution of the Lazarillo mRNA paralleled that of the monoclonal antibody labeling on embryos. Sequence analysis defines Lazarillo as a member of the lipocalin family, extracellular carriers of small hydrophobic ligands, and most related to the porphyrin- and retinol-binding lipocalins. Lazarillo is the first example of a lipocalin anchored to the plasma membrane, highly glycosylated, and restricted to a subset of developing neurons.Trabajo presentado al Annual Biomedical Research Conference for Minority Students celebrada en Nashville (US) del 13 al 16 de noviembre de 2013.A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATTs apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

The Bacillus subtilis 168 csn gene encodes a chitosanase with similar properties to a Streptomyces enzyme

The Bacillus subtilis 168 csn gene encodes a chitosanase. It was found that transcription of the csn gene was temporally regulated and was not subject to metabolic repression. Chitosanase synthesis was abolished in a csn mutant strain. Csn was overproduced in B. subtilis, partially purified and characterized. The deduced amino acid sequence, K(m), and optimal pH and temperature of the B. subtilis enzyme were closer to those of a chitosanase from Streptomyces sp. N174 than to those of chitosanases from other Bacillus strains.

In vivo transcription of bacteriophage φ29 DNA early and late promoter sequences

The in vivo transcription initiation sites of eight putative phi 29 promoters have been accurately determined: seven of them correspond to early promoters, including the four main ones, and the other corresponds to the only late promoter found in vivo. Comparison of the phi 29 promoter sequences with the consensus sequence for the Bacillus subtilis sigma 43 RNA polymerase suggests that the sigma 43 enzyme is involved in the recognition of the viral early promoters, whereas the late promoter sequences share homology with the consensus sequence only at its -10 region.

Streptomyces lividans groES, groEL1 and groEL2 genes

The Streptomyces lividans groES/EL1 operon and groEL2 gene were cloned and their respective DNA sequences determined. The sequenced DNA comprised the genes and their respective regulatory regions in both cases. Transcription of both groES/EL1 and groEL2 seemed to be subjected to temporal control at 30 degrees C. At 45 degrees C the amount of the groEL2 transcript increased considerably in comparison to that of groES/EL1. Among the proteins synthesized under heat shock by S. lividans, a fraction enriched in GroEL2 showed the presence of a ring-shaped structure that resembles that of other chaperonins and was active in a rhodanase folding assay.