Generation and analysis of expressed sequence tags from Botrytis cinerea

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000200018&lng=es&nrm=isoBotrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen

Predicción bioinformática y validación experimental de genes RNA regulatorios pequeños en la bactería extremófila Acidithiobacillus ferrooxidans

Tesis (Doctor en Biotecnología)RESUMEN: En bacterias los RNA reguladores pequeños (srRNA – “small regulatory RNA”) controlan la expresión génica, usualmente a nivel post-transcripcional. Esto lo hacen actuando como RNAs anti-sentido uniéndose al transcrito (mRNA) de los genes blanco (mRNAs) o interactuando con las proteínas reguladoras. Los srRNAs están involucrados en la regulación de una amplia variedad de procesos, tales como la replicación de plasmidios, el transporte, la replicación viral, la virulencia bacteriana y algunos de los circuitos genéticos globales que responden a cambios ambientales. Desde su descubrimiento, hace unos pocos años, se ha comprobado que se encuentran presentes en una variedad de organismos distintos. Sin embargo, su detección/predicción utilizando herramientas bioinformática es particularmente desafiante dado que no codifican para proteínas, son poco conservados a nivel nucleotídico y sólo algunos exhiben estructura secundaria conservada. En este trabajo utilizamos herramientas computacionales para predecir los srRNAs en la secuencia genómica de la bacteria extremófila Acidithiobacillus ferrooxidans, uno de los microorganismos más utilizados en la recuperación biológica de metales a partir de minerales azufrados, y herramientas de biología molecular para validarlos. La estrategia empleada involucra en primer lugar un análisis de genómica comparativa utilizando los genomas de Acidithiobacillus caldus y Acidithiobacillus thiooxidans (secuenciados por Fundación Ciencia para la Vida [88]) para identificar candidatos de srRNA conservados en las regiones intergénicas en bacterias cercanas filogenéticamente. Las regiones intergénicas conservadas fueron extraídas del genoma de A. ferrooxidans e investigadas para determinar cuáles de las secuencias son complementarias a genes que codifican proteínas y se asocian a promotores sigma 70 predichos. En segundo término, se estudió la expresión de los candidatos srRNAs por Northern blot y “RACE”. Como producto de este análisis se derivaron 11 nuevos srRNA en A. ferrooxidans. Adicionalmente, se ha desarrollado un nuevo programa para identificar posibles RNAs blancos de srRNA. Este programa, llamado “Kissing Complex”, se enfoca en la X búsqueda de conexiones anti-sentido entre srRNAs y mRNAs (RNAs blancos) en la región simple hebra, específicamente, donde el RNA forma lazos o “loops”. La identificación de los srRNAs potenciales entrega información relevante y novedosa en relación a la regulación de la expresión génica en A. ferrooxidans, permitiendo ampliar las áreas de investigación abordadas hasta la fecha y abriendo nuevas ventanas en los estudios biológicos de este microorganismo, contribuyendo así a nuestra comprensión del inusual metabolismo de las bacterias acidófilas y, eventualmente, a nuestro entendimiento de la biolixiviación.ABSTRACT: All bacteria contain small regulatory RNAs (srRNAs), ranging in size from 50 to 500 nucleotides, which control gene expression. srRNAs are proving to be multifunctional and have provided explanations for a number of previously mysterious regulatory effects. Phages and plasmids have long been recognized to use antisense RNA regulators but now sRNAs are being discovered in all bacterial genomes, including pathogens. In eukaryotic cells, microRNAs and RNAi parallel in many ways the bacterial srRNAs, confirming that this level of regulation is widespread. However, the computational discovery of srRNAs is particularly challenging since they do not encode protein products, are poorly conserved at the sequence level and only some exhibit conserved secondary structure. This thesis describes computational predictions and experimental validations of srRNAs in the chemolithoautotrophic acidophilic genera of bacteria termed "Acidithiobacilli". Acidithiobacilli are known to play an important role in the industrial process of metal recovery termed "bioleaching" or "biomining". I characterized the sequence and genomic features of these RNAs and incorporated the identified characteristics in a predictive scheme by employing the following principles: 1) I focused on intergenic regions, defined between annotated genes based on the A. ferrooxidans database. (2) Within these regions, I searched for transcription initiation and termination signals that are widely used by the E. coli transcription machinery and that were also observed in the known srRNAs genes. We focused on promoter DNA sequences recognized by the major RNA polymerase sigma factor, σ70, and on Rho-independent terminators, in which the termination signal resides in specific sequence and structural features of the RNA. (3) Among the predicted sequences, I chose those in which the distance between the predicted promoter and terminator was 50–500 base pairs. (4) The predicted sequences obtained were compared to genome sequences of other “Acidithiobacilli”, and those that showed significant conservation XII were selected. This screen resulted in the identification of 30 candidate srRNAs encoding genes. Experimental validation is presented for 11 novel srRNAs. Additionally, I developed a new computational program to identify potential target genes for predicted srRNA. This program, called "Kissing Complex", finds antisense connections between srRNAs and mRNA (target RNAs) in the single strand regions of both molecules, specifically, where the RNA forms loops. SrRNAs are known to control gene expression in a wide variety of microorganisms, usually at the post-transcriptional level, by acting as antisense RNAs that bind targeted mRNAs or by interacting with regulatory proteins. SrRNAs are involved in the regulation of a large variety of processes such as plasmid replication, transposition and global genetic circuits that respond to environmental changes. It is expected that future work aimed at the elucidation of the function of the srRNAs described herein will contribute to our understanding of the unusual metabolism of acidophilic bacteria and ultimately to our knowledge of bioleaching

Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium

<p>The genome of the acidophilic, bioleaching bacterium <i>Acidithiobacillus ferrooxidans</i>, strain ATCC 23270, contains 95 predicted tRNA genes. Thirty-six of these genes (all 20 species) are clustered within an actively excising integrative-conjugative element (ICEA<i>fe</i>1). We speculated that these tRNA genes might have a role in adapting the bacterial tRNA pool to the codon usage of ICEA<i>fe</i>1 genes. To answer this question, we performed theoretical calculations of the global tRNA adaptation index to the entire <i>A. ferrooxidans</i> genome with and without the ICEA<i>fe</i>1 encoded tRNA genes. Based on these calculations, we observed that tRNAs encoded in ICEA<i>fe</i>1 negatively contribute to adapt the tRNA pool to the codon use in <i>A. ferrooxidans.</i> Although some of the tRNAs encoded in ICE<i>Afe</i>1 are functional in aminoacylation or protein synthesis, we found that they are expressed at low levels. These findings, along with the identification of a tRNA-like RNA encoded in the same cluster, led us to speculate that tRNA genes encoded in the mobile genetic element ICEA<i>fe</i>1 might have acquired mutations that would result in either inactivation or the acquisition of new functions.</p

Architecture and gene repertoire of the flexible genome of the extreme acidophile Acidithiobacillus caldus

CITATION: Acuna, L. G. et al. 2013. Architecture and gene repertoire of the flexible genome of the extreme acidophile Acidithiobacillus caldus. PLoS ONE, 8(11):e78237, doi:10.1371/journal.pone.0078237.The original publication is available at http://journals.plos.orgBackground: Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome. Principal Findings: Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions. Significance: For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078237Publisher's versio

Diversity and abundance of IS families in <i>A. caldus</i> sequenced genomes.

<p>Bars represent the number of insertion sequence families predicted in the <i>A. caldus</i> type strain (white) and SM-1 strain (grey) genomes associated to the core and flexible gene complement. Lines represent the total number of sequences in each category in the <i>A. caldus</i> type strain (triangle) and SM-1 strain (square). Abbreviations: ICE, integrative conjugative element; IME, integrative mobilizable element; GI, genomic islands.</p

Functional categorization of <i>A. caldus</i> strain-specific genes.

<p>Bars represent the percentage of gene functions falling into the COG categories indicated. (A) Core genome, (B) Flexible genome of <i>A. caldus</i> ATCC 51756 (light grey) and <i>A. caldus</i> SM-1 (black). Genes with unknown functions were excluded from both graphs. These represent 35% of the core genome of the species and 64–71% of the flexible genome of <i>A. caldus</i> type strain and <i>A. caldus</i> SM-1, respectively. (a) Replication, recombination and repair; (b) Cell cycle control, cell division, chromosome partitioning; (c) Transcription; (d) Translation, ribosomal structure and biogenesis; (e) Posttranslational modification, protein turnover, chaperones; (f) Energy production and conversion; (g) Carbohydrate transport and metabolism; (h) Amino acid transport and metabolism; (i) Lipid transport and metabolism; (j) Nucleotide transport and metabolism; (k) Coenzyme transport and metabolism; (l) Inorganic ion transport and metabolism; (m) Secondary metabolites biosynthesis, transport and catabolism; (n) Cell wall/membrane/envelope biogenesis; (o) Cell motility; (p) Signal transduction mechanisms; (q) Intracellular trafficking, secretion, and vesicular transport; (r) Defense mechanisms.</p

Types of Integrative MGEs in <i>A. caldus</i> strains ATCC 51756 and SM-1.

(*)<p>ICE_Type 1 elements are >98% similar in about 56 Kb of their sequences;</p>(#)<p>ICE_Type 2 elements are >90% similar in about 100 Kb of their sequences,</p>(¢)<p>GI_Type 1 elements are almost identical,</p>(&)<p>GI_Type 5 elements are almost identical.</p><p>Abbreviations: ICE, Integrative Conjugative Element; GI, Genomic Island; IME, Integrative Movilizable Element; Int, Integrase; Int(t), Truncated Integrase; Xis, Excisionase; cI, CI-family phage regulator, cII, CII-family phage regulator; Cro, CRO-family phage regulator; T4SS, Type IV secretion system.</p

Genomic comparison of <i>A. caldus</i> strains ATCC 51756 and SM-1.

<p>Similarities and differences between sequenced <i>A. caldus</i> strains revealed by <i>in silico</i> analysis of (A) the chromosome and (B) the megaplasmids. Chromosomes are represented using ACT. Features are color-coded as follows, grey, CDS or coding sequences; light purple, transposases; light orange tRNAs; light green, rRNA; red, prophage <i>Aca</i>ML1; black, ICE or integrative conjugative element; orange, IME or integrative mobilizable element; green, GI or genomic island; purple, ISR or IS rich regions; white, conserved gene modules numbered according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078237#pone.0078237.s003" target="_blank">Table S3a</a>.</p