RESUMO
Analyzing transcription data requires intensive statistical analysis to obtain useful biological information and knowledge. A significant portion of this data is affected by random noise or even noise intrinsic to the modeling of the experiment. Without robust treatment, the data might not be explored thoroughly, and incorrect conclusions could be drawn. Examining the correlation between gene expression profiles is one way bioinformaticians extract information from transcriptomic experiments. However, the correlation measurements traditionally used have worrisome shortcomings that need to be addressed. This paper compares five already published and experimented-with correlation measurements to the newly developed coincidence index, a similarity measurement that combines Jaccard and interiority indexes and generalizes them to be applied to vectors containing real values. We used microarray and RNA-Seq data from the archaeonHalobacterium salinarumand the bacteriumEscherichia coli, respectively, to evaluate the capacity of each correlation/similarity measurement. The utilized method explores the co-expressed metabolic pathways by measuring the correlations between the expression levels of enzymes that share metabolites, represented in the form of a weighted graph. It then searches for local maxima in this graph using a simulated annealing algorithm. We demonstrate that the coincidence index extracts larger, more comprehensive, and more statistically significant pathways for microarray experiments. In RNA-Seq experiments, the results are more limited, but the coincidence index managed the largest percentage of significant components in the graph.
Assuntos
Escherichia coli , Halobacterium salinarum , Redes e Vias Metabólicas , Escherichia coli/genética , Escherichia coli/metabolismo , Halobacterium salinarum/metabolismo , Halobacterium salinarum/genética , Perfilação da Expressão Gênica/métodos , Biologia Computacional , Transcriptoma , Algoritmos , RNA-SeqRESUMO
Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.
Assuntos
Carboxilesterase , Clonagem Molecular , Halobacterium salinarum , Proteínas Recombinantes , Carboxilesterase/genética , Carboxilesterase/metabolismo , Carboxilesterase/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Halobacterium salinarum/enzimologia , Halobacterium salinarum/genética , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Concentração de Íons de Hidrogênio , Cinética , Estabilidade Enzimática , Proteínas Arqueais/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , TemperaturaRESUMO
Post-transcriptional processing of messenger RNA is an important regulatory strategy that allows relatively fast responses to changes in environmental conditions. In halophile systems biology, the protein perspective of this problem (i.e., ribonucleases which implement the cleavages) is generally more studied than the RNA perspective (i.e., processing sites). In the present in silico work, we mapped genome-wide transcriptional processing sites (TPS) in two halophilic model organisms, Halobacterium salinarum NRC-1 and Haloferax volcanii DS2. TPS were established by reanalysis of publicly available differential RNA-seq (dRNA-seq) data, searching for non-primary (monophosphorylated RNAs) enrichment. We found 2093 TPS in 43% of H. salinarum genes and 3515 TPS in 49% of H. volcanii chromosomal genes. Of the 244 conserved TPS sites found, the majority were located around start and stop codons of orthologous genes. Specific genes are highlighted when discussing antisense, ribosome and insertion sequence associated TPS. Examples include the cell division gene ftsZ2, whose differential processing signal along growth was detected and correlated with post-transcriptional regulation, and biogenesis of sense overlapping transcripts associated with IS200/IS605. We hereby present the comparative, transcriptomics-based processing site maps with a companion browsing interface.
Assuntos
Proteínas Arqueais/genética , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Halobacterium salinarum/genética , Haloferax volcanii/genética , Sítio de Iniciação de Transcrição , Transcriptoma , Proteínas Arqueais/metabolismo , Halobacterium salinarum/metabolismo , Haloferax volcanii/metabolismo , RNA-Seq , RibossomosRESUMO
Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3' ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxinâ»antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea.
Assuntos
Perfilação da Expressão Gênica , Halobacterium salinarum/genética , RNA Antissenso/genética , Transcriptoma/genética , Regulação da Expressão Gênica em Archaea/genética , Genoma Arqueal/genética , RNA não Traduzido/genética , Ribossomos/genética , Sítio de Iniciação de TranscriçãoRESUMO
Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.
Assuntos
Processamento Alternativo , Proteínas Arqueais/metabolismo , Genoma Arqueal , Halobacterium salinarum/metabolismo , Fases de Leitura Aberta , RNA Antissenso/genética , RNA Arqueal/genética , Proteínas Arqueais/genética , Sequência de Bases , Perfilação da Expressão Gênica , Halobacterium salinarum/genética , Halobacterium salinarum/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Sítio de Iniciação de TranscriçãoRESUMO
Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli's specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.
Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Halobacterium salinarum/genética , Clonagem Molecular/métodos , DNA Arqueal , Tamanho do Genoma , Modelos GenéticosRESUMO
The existence of sense overlapping transcripts that share regulatory and coding information in the same genomic sequence shows an additional level of prokaryotic gene expression complexity. Here we report the discovery of ncRNAs associated with IS1341-type transposase (tnpB) genes, at the 3'-end of such elements, with examples in archaea and bacteria. Focusing on the model haloarchaeon Halobacterium salinarum NRC-1, we show the existence of sense overlapping transcripts (sotRNAs) for all its IS1341-type transposases. Publicly available transcriptome compendium show condition-dependent differential regulation between sotRNAs and their cognate genes. These sotRNAs allowed us to find a UUCA tetraloop motif that is present in other archaea (ncRNA family HgcC) and in a H. salinarum intergenic ncRNA derived from a palindrome associated transposable elements (PATE). Overexpression of one sotRNA and the PATE-derived RNA harboring the tetraloop motif improved H. salinarum growth, indicating that these ncRNAs are functional.
Assuntos
Genes Arqueais , Halobacterium salinarum/genética , RNA não Traduzido/genética , Transposases/genética , Sequência de Bases , Perfilação da Expressão Gênica , Halobacterium salinarum/crescimento & desenvolvimento , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retroelementos/genéticaRESUMO
A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (â¼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.
Assuntos
Archaea/genética , RNA Arqueal/fisiologia , RNA não Traduzido/fisiologia , Regulação da Expressão Gênica em Archaea , Halobacterium salinarum/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mathanococcus/genética , Pyrococcus furiosus/genética , Análise de Sequência de RNA , Sulfolobus solfataricus/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica , TranscriptomaRESUMO
Propidium monoazide (PMA) is a DNA-intercalating agent used to selectively detect DNA from viable cells by polymerase chain reaction (PCR). Here, we report that high concentrations (>5%) of sodium chloride (NaCl) prevents PMA from inhibiting DNA amplification from dead cells. Moreover, Halobacterium salinarum was unable to maintain cell integrity in solutions containing less than 15% NaCl, indicating that extreme halophilic microorganisms may not resist the concentration range in which PMA fully acts. We conclude that NaCl, but not pH, directly affects the efficiency of PMA treatment, limiting its use for cell viability assessment of halophiles and in hypersaline samples.
Assuntos
Azidas/farmacologia , Halobacterium salinarum/citologia , Halobacterium salinarum/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Propídio/análogos & derivados , Cloreto de Sódio/farmacologia , DNA Bacteriano/genética , Genoma Bacteriano/genética , Halobacterium salinarum/genética , Reação em Cadeia da Polimerase , Propídio/farmacologiaRESUMO
The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA(Asn) formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA(Asn). The misacylated tRNA is then converted to Asn-tRNA(Asn) by the action of an Asp-tRNA(Asn) amidotransferase. Here we show that Asn-tRNA(Asn) formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA(Asn) in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA(Asn) genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA(Asn). The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA(Asn). Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.