Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration... more Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration. In spite of being an established model microorganism for the archaea domain, direct comparisons between its proteome and transcriptome during osmotic stress are still not available. Through RNA-seq-based transcriptomics, we compared a low salt (2.6 M NaCl) stress condition with 4.3 M of NaCl and found 283 differentially expressed loci. The more commonly found classes of genes were: ABC-type transporters and transcription factors. Similarities, and most importantly, differences between our findings and previously published datasets in similar experimental conditions are discussed. We validated three important biological processes differentially expressed: gas vesicles production (due to down-regulation of gvpA1b, gvpC1b, gvpN1b, and gvpO1b); archaellum formation (due to down-regulation of arlI, arlB1, arlB2, and arlB3); and glycerol metabolism (due to up-regulation of glpA1, glpB, and g...
ABSTRACTThe scale of post-transcriptional regulation and the implications of its interplay with o... more ABSTRACTThe scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation on environmental acclimation is underexplored for organisms of the domain Archaea. Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating transcriptome-wide locations of transcript processing sites (TPS) and SmAP1 binding, genome-wide locations of antisense RNAs (asRNAs), and consequences of RNase_2099C knockout on differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (RNA-Seq) and protein levels (SWATH-MS) for 2,579 genes over ...
The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide vari... more The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). ABSTRACT The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.
Background Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare ... more Background Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. Results Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor Ohr...
Low-temperature stress is an important factor for nucleic acid stability and must be circumvented... more Low-temperature stress is an important factor for nucleic acid stability and must be circumvented in order to maintain the basic cell processes, such as transcription and translation. The oligotrophic lifestyle presents further challenges to ensure the proper nutrient uptake and osmotic balance in an environment of slow nutrient flow.
Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulat... more 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 toxin–antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found ...
Prokaryotic genomes show a high level of information compaction often with different molecules tr... more 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 visicle protein gene g...
Our ability to genetically manipulate living organisms is usually constrained by the efficiency o... more 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.
Genetics and molecular research : GMR, Jan 31, 2006
SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely av... more SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella f...
The existence of sense overlapping transcripts that share regulatory and coding information in th... more 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, indicat...
A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, in... more 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 (,7% 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.
Background: The search for enriched (aka over-represented or enhanced) ontology terms in a list o... more Background: The search for enriched (aka over-represented or enhanced) ontology terms in a list of genes obtained from microarray experiments is becoming a standard procedure for a systemlevel analysis. This procedure tries to summarize the information focussing on classification designs such as Gene Ontology, KEGG pathways, and so on, instead of focussing on individual genes. Although it is well known in statistics that association and significance are distinct concepts, only the former approach has been used to deal with the ontology term enrichment problem. Results: BayGO implements a Bayesian approach to search for enriched terms from microarray data. The R source-code is freely available at http://blasto.iq.usp.br/~tkoide/BayGO in three versions: Linux, which can be easily incorporated into pre-existent pipelines; Windows, to be controlled interactively; and as a web-tool. The software was validated using a bacterial heat shock response dataset, since this stress triggers known system-level responses. Conclusion: The Bayesian model accounts for the fact that, eventually, not all the genes from a given category are observable in microarray data due to low intensity signal, quality filters, genes that were not spotted and so on. Moreover, BayGO allows one to measure the statistical association between generic ontology terms and differential expression, instead of working only with the common significance analysis.
Technologies to synthesize and transplant a complete genome into a cell have opened limitless pot... more Technologies to synthesize and transplant a complete genome into a cell have opened limitless potential to redesign organisms for complex, specialized tasks. However, large-scale re-engineering of a biological circuit will require systems-level optimization that will come from a deep understanding of operational relationships among all the constituent parts of a cell. The integrated framework necessary for conducting such complex bioengineering requires the convergence of systems and synthetic biology. Here, we review the status of these rapidly developing interdisciplinary fields of biology and provide a perspective on plausible venues for their merger. Multidisciplinary approaches in science have always resulted in new ideas, broader and deeper levels of understanding, and of course, controversies. The inception of many modern transformative products and technologies, including drug synthesis 1 and recombinant microbial enzyme factories 2 can be attributed to the merging of, until then, distinct disciplines. With recent advances to deconstruct biological function at a systems scale 3,4 and design biological subcircuits with defined properties 5 we can begin to envision spectacular solutions that combine unique functions that have evolved in organisms from diverse environments. In other words, the stage is set for yet another revolutionary merger of two powerful interdisciplinary fields with fundamentally different but complementary outlooks-synthetic biology and systems biology (FIG. 1). A systems biologist aims to model and understand an entire organism by characterizing dynamic environment-dependent functional interrelationships between its constituent parts (for example, genes, RNAs, proteins and metabolites). A synthetic biologist, however, uses well characterized parts that are shaped by natural evolution to construct artificial systems that perform new tasks. These fields are on trajectories that are bound to cross paths and even merge as they begin to inform one another. We envision that systems biology will provide both the
During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer ... more During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC-1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis-regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino-acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB-regulated metabolic network includes enzyme-coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co-evolution in which TrmB integrates the expression of metabolic enzyme-coding genes of diverse origins. Molecular Systems Biology 5: 282
Background: Identification of SCF1(FBXO25) substrates can be done through in chip ubiquitination ... more Background: Identification of SCF1(FBXO25) substrates can be done through in chip ubiquitination on protoarrays. Results: FBXO25 interacts and mediates ubiquitination and proteasomal degradation of the ELK-1 protooncogene regulator. Conclusion: The c-Fos regulator ELK-1 is an SCF1(FBXO25) substrate. Significance: FBXO25 is a potential mitogen pathway regulator through ELK-1 degradation. FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.
Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, ... more Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA , predicted to encode an e xtra c ytoplasmic f unction (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa σ E regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 σ E -dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative σ E -dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like othe...
As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, c... more As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella. Vector pSP3 was constructed by ligating to the E. coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3. Transformation experiments have shown that pSP3 replicates stably in Xylella. When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture. These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration... more Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration. In spite of being an established model microorganism for the archaea domain, direct comparisons between its proteome and transcriptome during osmotic stress are still not available. Through RNA-seq-based transcriptomics, we compared a low salt (2.6 M NaCl) stress condition with 4.3 M of NaCl and found 283 differentially expressed loci. The more commonly found classes of genes were: ABC-type transporters and transcription factors. Similarities, and most importantly, differences between our findings and previously published datasets in similar experimental conditions are discussed. We validated three important biological processes differentially expressed: gas vesicles production (due to down-regulation of gvpA1b, gvpC1b, gvpN1b, and gvpO1b); archaellum formation (due to down-regulation of arlI, arlB1, arlB2, and arlB3); and glycerol metabolism (due to up-regulation of glpA1, glpB, and g...
ABSTRACTThe scale of post-transcriptional regulation and the implications of its interplay with o... more ABSTRACTThe scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation on environmental acclimation is underexplored for organisms of the domain Archaea. Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating transcriptome-wide locations of transcript processing sites (TPS) and SmAP1 binding, genome-wide locations of antisense RNAs (asRNAs), and consequences of RNase_2099C knockout on differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (RNA-Seq) and protein levels (SWATH-MS) for 2,579 genes over ...
The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide vari... more The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). ABSTRACT The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.
Background Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare ... more Background Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. Results Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor Ohr...
Low-temperature stress is an important factor for nucleic acid stability and must be circumvented... more Low-temperature stress is an important factor for nucleic acid stability and must be circumvented in order to maintain the basic cell processes, such as transcription and translation. The oligotrophic lifestyle presents further challenges to ensure the proper nutrient uptake and osmotic balance in an environment of slow nutrient flow.
Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulat... more 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 toxin–antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found ...
Prokaryotic genomes show a high level of information compaction often with different molecules tr... more 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 visicle protein gene g...
Our ability to genetically manipulate living organisms is usually constrained by the efficiency o... more 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.
Genetics and molecular research : GMR, Jan 31, 2006
SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely av... more SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella f...
The existence of sense overlapping transcripts that share regulatory and coding information in th... more 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, indicat...
A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, in... more 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 (,7% 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.
Background: The search for enriched (aka over-represented or enhanced) ontology terms in a list o... more Background: The search for enriched (aka over-represented or enhanced) ontology terms in a list of genes obtained from microarray experiments is becoming a standard procedure for a systemlevel analysis. This procedure tries to summarize the information focussing on classification designs such as Gene Ontology, KEGG pathways, and so on, instead of focussing on individual genes. Although it is well known in statistics that association and significance are distinct concepts, only the former approach has been used to deal with the ontology term enrichment problem. Results: BayGO implements a Bayesian approach to search for enriched terms from microarray data. The R source-code is freely available at http://blasto.iq.usp.br/~tkoide/BayGO in three versions: Linux, which can be easily incorporated into pre-existent pipelines; Windows, to be controlled interactively; and as a web-tool. The software was validated using a bacterial heat shock response dataset, since this stress triggers known system-level responses. Conclusion: The Bayesian model accounts for the fact that, eventually, not all the genes from a given category are observable in microarray data due to low intensity signal, quality filters, genes that were not spotted and so on. Moreover, BayGO allows one to measure the statistical association between generic ontology terms and differential expression, instead of working only with the common significance analysis.
Technologies to synthesize and transplant a complete genome into a cell have opened limitless pot... more Technologies to synthesize and transplant a complete genome into a cell have opened limitless potential to redesign organisms for complex, specialized tasks. However, large-scale re-engineering of a biological circuit will require systems-level optimization that will come from a deep understanding of operational relationships among all the constituent parts of a cell. The integrated framework necessary for conducting such complex bioengineering requires the convergence of systems and synthetic biology. Here, we review the status of these rapidly developing interdisciplinary fields of biology and provide a perspective on plausible venues for their merger. Multidisciplinary approaches in science have always resulted in new ideas, broader and deeper levels of understanding, and of course, controversies. The inception of many modern transformative products and technologies, including drug synthesis 1 and recombinant microbial enzyme factories 2 can be attributed to the merging of, until then, distinct disciplines. With recent advances to deconstruct biological function at a systems scale 3,4 and design biological subcircuits with defined properties 5 we can begin to envision spectacular solutions that combine unique functions that have evolved in organisms from diverse environments. In other words, the stage is set for yet another revolutionary merger of two powerful interdisciplinary fields with fundamentally different but complementary outlooks-synthetic biology and systems biology (FIG. 1). A systems biologist aims to model and understand an entire organism by characterizing dynamic environment-dependent functional interrelationships between its constituent parts (for example, genes, RNAs, proteins and metabolites). A synthetic biologist, however, uses well characterized parts that are shaped by natural evolution to construct artificial systems that perform new tasks. These fields are on trajectories that are bound to cross paths and even merge as they begin to inform one another. We envision that systems biology will provide both the
During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer ... more During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC-1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis-regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino-acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB-regulated metabolic network includes enzyme-coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co-evolution in which TrmB integrates the expression of metabolic enzyme-coding genes of diverse origins. Molecular Systems Biology 5: 282
Background: Identification of SCF1(FBXO25) substrates can be done through in chip ubiquitination ... more Background: Identification of SCF1(FBXO25) substrates can be done through in chip ubiquitination on protoarrays. Results: FBXO25 interacts and mediates ubiquitination and proteasomal degradation of the ELK-1 protooncogene regulator. Conclusion: The c-Fos regulator ELK-1 is an SCF1(FBXO25) substrate. Significance: FBXO25 is a potential mitogen pathway regulator through ELK-1 degradation. FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.
Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, ... more Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA , predicted to encode an e xtra c ytoplasmic f unction (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa σ E regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 σ E -dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative σ E -dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like othe...
As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, c... more As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella. Vector pSP3 was constructed by ligating to the E. coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3. Transformation experiments have shown that pSP3 replicates stably in Xylella. When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture. These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
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Papers by Tie Koide