RESUMEN
The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Filogenia , Factores de Transcripción/genética , Escherichia coli/genética , Escherichia coli/metabolismo , División Celular/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismoRESUMEN
Synthetic small RNAs (sRNAs) are gaining increasing attention in the field of synthetic biology and bioengineering for efficient post-transcriptional regulation of gene expression. However, the optimal design of synthetic sRNAs is challenging because alterations may impair functions or off-target effects can arise. Here, we introduce DIGGER-Bac, a toolbox for Design and Identification of seed regions for Golden Gate assembly and Expression of synthetic sRNAs in Bacteria. The SEEDling tool predicts optimal sRNA seed regions in combination with user-defined sRNA scaffolds for efficient regulation of specified mRNA targets. Results are passed on to the G-GArden tool, which assists with primer design for high-fidelity Golden Gate assembly of the desired synthetic sRNA constructs.
Asunto(s)
ARN Bacteriano , ARN Pequeño no Traducido , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Bacterias/genética , Bacterias/metabolismo , ARN Mensajero/genética , ARN Pequeño no Traducido/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS-induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read-through transcription into a cryptic prophage encoding kilR The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS-mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Proteínas Represoras/genética , Proteínas Bacterianas/genética , División Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Inestabilidad Genómica/genética , Estrés Oxidativo/fisiología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis.plantcell;31/12/2912/FX1F1fx1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Metaloproteasas/metabolismo , Nutrientes/deficiencia , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas Represoras/metabolismo , Synechocystis/metabolismo , Aclimatación/genética , Proteínas Bacterianas/genética , Carbono/deficiencia , Carbono/metabolismo , Expresión Génica , Metaloproteasas/genética , Mutación , Nitrógeno/deficiencia , Nitrógeno/metabolismo , Nutrientes/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Fosfatos/deficiencia , Fosfatos/metabolismo , Fosforilación , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/genética , Proteolisis , Proteoma/genética , Proteoma/metabolismo , Proteómica , Regulón/genética , Proteínas Represoras/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Synechocystis/enzimología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic microRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNAs targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB, and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more details. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable.
Asunto(s)
Biología Computacional/métodos , Regulación Bacteriana de la Expresión Génica/genética , ARN Pequeño no Traducido/genética , Bacterias/genética , Enterobacteriaceae/genética , Expresión Génica/genética , Genes Bacterianos/genética , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Staphylococcus aureus/genéticaRESUMEN
MOTIVATION: The correct prediction of bacterial sRNA homologs is a prerequisite for many downstream analyses based on comparative genomics, but it is frequently challenging due to the short length and distinct heterogeneity of such homologs. GLobal Automatic Small RNA Search go (GLASSgo) is an efficient tool for the prediction of sRNA homologs from a single input query. To make the algorithm available to a broader community, we offer a Docker container along with a free-access web service. For non-computer scientists, the web service provides a user-friendly interface. However, capabilities were lacking so far for batch processing, version control and direct interaction with compatible software applications as a workflow management system can provide. RESULTS: Here, we present GLASSgo 1.5.2, an updated version that is fully incorporated into the workflow management system Galaxy. The improved version contains a new feature for extracting the upstream regions, allowing the search for conserved promoter elements. Additionally, it supports the use of accession numbers instead of the outdated GI numbers, which widens the applicability of the tool. AVAILABILITY AND IMPLEMENTATION: GLASSgo is available at https://github.com/lotts/GLASSgo/ under the MIT license and is accompanied by instruction and application data. Furthermore, it can be installed into any Galaxy instance using the Galaxy ToolShed.
Asunto(s)
Biología Computacional , Programas Informáticos , Algoritmos , Genómica , Flujo de TrabajoRESUMEN
DnaA is the initiator protein of chromosome replication, but the regulation of its homoeostasis in enterobacteria is not well understood. The DnaA level remains stable at different growth rates, suggesting a link between metabolism and dnaA expression. In a bioinformatic prediction, which we made to unravel targets of the sRNA rnTrpL in Enterobacteriaceae, the dnaA mRNA was the most conserved target candidate. The sRNA rnTrpL is derived from the transcription attenuator of the tryptophan biosynthesis operon. In Escherichia coli, its level is higher in minimal than in rich medium due to derepressed transcription without external tryptophan supply. Overexpression and deletion of the rnTrpL gene decreased and increased, respectively, the levels of dnaA mRNA. The decrease of the dnaA mRNA level upon rnTrpL overproduction was dependent on hfq and rne. Base pairing between rnTrpL and dnaA mRNA in vivo was validated. In minimal medium, the oriC level was increased in the ΔtrpL mutant, in line with the expected DnaA overproduction and increased initiation of chromosome replication. In line with this, chromosomal rnTrpL mutation abolishing the interaction with dnaA increased both the dnaA mRNA and the oriC level. Moreover, upon addition of tryptophan to minimal medium cultures, the oriC level in the wild type was increased. Thus, rnTrpL is a base-pairing sRNA that posttranscriptionally regulates dnaA in E. coli. Furthermore, our data suggest that rnTrpL contributes to the DnaA homoeostasis in dependence on the nutrient availability, which is represented by the tryptophan level in the cell.
Asunto(s)
Proteínas Bacterianas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Operón , ARN Pequeño no Traducido/metabolismo , Transcripción Genética , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , ARN Pequeño no Traducido/genéticaRESUMEN
The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de.
Asunto(s)
Secuencia de Bases/genética , Internet , ARN/genética , Programas Informáticos , Algoritmos , Conformación de Ácido Nucleico , ARN/química , Alineación de Secuencia/instrumentación , Análisis de Secuencia de ARN/instrumentación , Relación Estructura-ActividadRESUMEN
Photosynthetic microorganisms encounter an erratic nutrient environment characterized by periods of iron limitation and sufficiency. Surviving in such an environment requires mechanisms for handling these transitions. Our study identified a regulatory system involved in the process of recovery from iron limitation in cyanobacteria. We set out to study the role of bacterioferritin co-migratory proteins during transitions in iron bioavailability in the cyanobacterium Synechocystis sp. PCC 6803 using knockout strains coupled with physiological and biochemical measurements. One of the mutants displayed slow recovery from iron limitation. However, we discovered that the cause of the phenotype was not the intended knockout but rather the serendipitous selection of a mutation in an unrelated locus, slr1658. Bioinformatics analysis suggested similarities to two-component systems and a possible regulatory role. Transcriptomic analysis of the recovery from iron limitation showed that the slr1658 mutation had an extensive effect on the expression of genes encoding regulatory proteins, proteins involved in the remodeling and degradation of the photosynthetic apparatus and proteins modulating electron transport. Most significantly, expression of the cyanobacterial homologue of the cyclic electron transport protein PGR5 was upregulated 1000-fold in slr1658 disruption mutants. pgr5 transcripts in the Δslr1658 mutant retained these high levels under a range of stress and recovery conditions. The results suggest that slr1658 is part of a regulatory operon that, among other aspects, affects the regulation of alternative electron flow. Disruption of its function has deleterious results under oxidative stress promoting conditions.
Asunto(s)
Proteínas Bacterianas/genética , Grupo Citocromo b/genética , Ferritinas/genética , Redes Reguladoras de Genes , Genoma Bacteriano/genética , Deficiencias de Hierro , Synechocystis/genética , Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Transporte de Electrón , Ferritinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Homeostasis , Hierro/metabolismo , Modelos Biológicos , Mutación , Operón/genética , Estrés Oxidativo , Fenotipo , Fotosíntesis , Synechocystis/crecimiento & desarrollo , Synechocystis/fisiología , Secuenciación Completa del GenomaRESUMEN
Nitrogen is frequently limiting microbial growth in the environment. As a response, many filamentous cyanobacteria differentiate heterocysts, cells devoted to N2 fixation. Heterocyst differentiation is under the control of the master regulator HetR. Through the characterization of the HetR-dependent transcriptome in Nostoc sp. PCC 7120, we identified the new candidate genes likely involved in heterocyst differentiation. According to their maximum induction, we defined E-DIF (early in differentiation) and L-DIF (late in differentiation) genes. Most of the genes known to be involved in the critical aspects of heterocyst differentiation or function were also classified into these groups, showing the validity of the approach. Using fusions to gfp, we verified the heterocyst-specific transcription of several of the found genes, antisense transcripts and potentially trans-acting sRNAs. Through comparative sequence analysis of promoter regions, we noticed the prevalence of the previously described DIF1 motif and identified a second motif, called DIF2, in other promoters of the E-DIF cluster. Both motifs are widely conserved in heterocystous cyanobacteria. We assigned alr2522 as a third member, besides nifB and nifP, to the CnfR regulon. The elements identified here are of interest for understanding cell differentiation, engineering of biological nitrogen fixation or production of O2 -sensitive molecules in cyanobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Nostoc/crecimiento & desarrollo , Nostoc/metabolismo , Transcriptoma , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Fijación del Nitrógeno , Nostoc/genética , Regiones Promotoras GenéticasRESUMEN
DEAD-box RNA-helicases catalyze the reorganization of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083), whose expression is regulated by abiotic stresses that alter the redox potential of the photosynthetic electron transport chain, including temperature downshift. Despite its proposed effect on RNA metabolism and its known relevance in cold-stress adaptation, the reported impact of a CrhR knockout on the cold adaption of the transcriptome only identified eight affected genes. Here, we utilized a custom designed microarray to assess the impact of the absence of CrhR RNA helicase activity on the transcriptome, independent of cold stress. CrhR truncation impacts an RNA subset comprising ~10% of the ncRNA and also ~10% of the mRNA transcripts. While equal numbers of mRNAs showed increased as well as decreased abundance, more than 90% of the ncRNAs showed enhanced expression in the absence of CrhR, indicative of a negative effect on ncRNA transcription or stability. We further tested the effect of CrhR on the stability of strongly responding RNAs that identify examples of post-transcriptional and transcriptional regulation. The data suggest that CrhR impacts multiple aspects of RNA metabolism in Synechocystis.
Asunto(s)
ARN Helicasas/metabolismo , Synechocystis/enzimología , Synechocystis/genética , Transcriptoma/genética , Activación Enzimática , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Semivida , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5'UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus [Formula: see text] assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Fijación del Nitrógeno/fisiología , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Synechocystis/fisiología , Factores de Transcripción/metabolismo , Northern Blotting , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Immunoblotting , Análisis por Micromatrices , Mutagénesis Sitio-Dirigida , Secuencias Reguladoras de Ácido Ribonucleico/genéticaRESUMEN
BACKGROUND: The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles. RESULTS: Physiological characterization of a 6S RNA deletion strain (ΔssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation are accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in ΔssaA. In vivo pull-down studies of the RNA polymerase complex indicated that the presence of 6S RNA promotes the recruitment of the cyanobacterial housekeeping σ factor SigA, concurrently supporting dissociation of group 2 σ factors during recovery from nitrogen starvation. CONCLUSIONS: The combination of genetic, physiological and biochemical studies reveals the homologue of 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by accelerating the switch from group 2 σ factors SigB, SigC and SigE to SigA-dependent transcription. We therefore introduce the cyanobacterial 6S RNA as a novel candidate regulator of RNA polymerase sigma factor recruitment in Synechocystis sp. PCC 6803. Further studies on mechanistic features of the postulated interaction should shed additional light on the complexity of transcriptional regulation in cyanobacteria.
Asunto(s)
Aclimatación/genética , Regulación Bacteriana de la Expresión Génica , Nitrógeno/deficiencia , ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Perfilación de la Expresión Génica , Fotosíntesis/genética , Ficobilisomas/genética , ARN Bacteriano/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , Factor sigma/metabolismo , Transactivadores/genéticaRESUMEN
Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.
Asunto(s)
Fotosíntesis , ARN Bacteriano/metabolismo , Synechocystis/metabolismo , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Endorribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genes Reporteros , Semivida , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Filogenia , Unión Proteica/genética , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Synechocystis/genética , Transcripción GenéticaRESUMEN
Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) have been suggested to be the two most critical factors for this response in Synechocystis sp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of the psrR1 gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence of psrR1 in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and the psrR1 gene is repressed by the binding of RpaB under low-light (LL) conditions. A decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression of psrR1 gene expression. Accumulating PsrR1 molecules then prevent translation from pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Complejo de Proteína del Fotosistema I/genética , Synechocystis/fisiología , Aclimatación/genética , Sitios de Unión , Retroalimentación Fisiológica , Luz , Complejo de Proteína del Fotosistema I/metabolismo , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Synechocystis/genéticaRESUMEN
Carbohydrate metabolism is a tightly regulated process in photosynthetic organisms. In the cyanobacterium Synechocystis sp. PCC 6803, the photomixotrophic growth protein A (PmgA) is involved in the regulation of glucose and storage carbohydrate (i.e. glycogen) metabolism, while its biochemical activity and possible factors acting downstream of PmgA are unknown. Here, a genome-wide microarray analysis of a ΔpmgA strain identified the expression of 36 protein-coding genes and 42 non-coding transcripts as significantly altered. From these, the non-coding RNA Ncr0700 was identified as the transcript most strongly reduced in abundance. Ncr0700 is widely conserved among cyanobacteria. In Synechocystis its expression is inversely correlated with light intensity. Similarly to a ΔpmgA mutant, a Δncr0700 deletion strain showed an approximately 2-fold increase in glycogen content under photoautotrophic conditions and wild-type-like growth. Moreover, its growth was arrested by 38 h after a shift to photomixotrophic conditions. Ectopic expression of Ncr0700 in Δncr0700 and ΔpmgA restored the glycogen content and photomixotrophic growth to wild-type levels. These results indicate that Ncr0700 is required for photomixotrophic growth and the regulation of glycogen accumulation, and acts downstream of PmgA. Hence Ncr0700 is renamed here as PmgR1 for photomixotrophic growth RNA 1.
Asunto(s)
Glucógeno/metabolismo , Procesos Fototróficos/genética , ARN no Traducido/metabolismo , Synechocystis/crecimiento & desarrollo , Synechocystis/genética , Secuencia de Bases , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Genoma Bacteriano , Genotipo , Luz , Mutación/genética , Procesos Fototróficos/efectos de la radiación , ARN no Traducido/genética , Reproducibilidad de los Resultados , Alineación de Secuencia , Synechocystis/efectos de la radiación , Transcripción Genética/efectos de la radiación , Regulación hacia Arriba/genéticaRESUMEN
CopraRNA (Comparative prediction algorithm for small RNA targets) is the most recent asset to the Freiburg RNA Tools webserver. It incorporates and extends the functionality of the existing tool IntaRNA (Interacting RNAs) in order to predict targets, interaction domains and consequently the regulatory networks of bacterial small RNA molecules. The CopraRNA prediction results are accompanied by extensive postprocessing methods such as functional enrichment analysis and visualization of interacting regions. Here, we introduce the functionality of the CopraRNA and IntaRNA webservers and give detailed explanations on their postprocessing functionalities. Both tools are freely accessible at http://rna.informatik.uni-freiburg.de.
Asunto(s)
ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/metabolismo , Programas Informáticos , Algoritmos , Redes Reguladoras de Genes , Internet , Análisis de Secuencia de ARNRESUMEN
Small RNAs (sRNAs) constitute a large and heterogeneous class of bacterial gene expression regulators. Much like eukaryotic microRNAs, these sRNAs typically target multiple mRNAs through short seed pairing, thereby acting as global posttranscriptional regulators. In some bacteria, evidence for hundreds to possibly more than 1,000 different sRNAs has been obtained by transcriptome sequencing. However, the experimental identification of possible targets and, therefore, their confirmation as functional regulators of gene expression has remained laborious. Here, we present a strategy that integrates phylogenetic information to predict sRNA targets at the genomic scale and reconstructs regulatory networks upon functional enrichment and network analysis (CopraRNA, for Comparative Prediction Algorithm for sRNA Targets). Furthermore, CopraRNA precisely predicts the sRNA domains for target recognition and interaction. When applied to several model sRNAs, CopraRNA revealed additional targets and functions for the sRNAs CyaR, FnrS, RybB, RyhB, SgrS, and Spot42. Moreover, the mRNAs gdhA, lrp, marA, nagZ, ptsI, sdhA, and yobF-cspC were suggested as regulatory hubs targeted by up to seven different sRNAs. The verification of many previously undetected targets by CopraRNA, even for extensively investigated sRNAs, demonstrates its advantages and shows that CopraRNA-based analyses can compete with experimental target prediction approaches. A Web interface allows high-confidence target prediction and efficient classification of bacterial sRNAs.
Asunto(s)
ARN Bacteriano/genética , Algoritmos , Secuencia de Bases , Biología Computacional , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Escherichia coli/clasificación , Escherichia coli/genética , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Genómica/estadística & datos numéricos , Filogenia , ARN Bacteriano/química , ARN Bacteriano/clasificación , Salmonella enterica/clasificación , Salmonella enterica/genéticaRESUMEN
Iron and manganese are part of a small group of transition metals required for photosynthetic electron transport. Here, we present evidence for a functional link between iron and manganese homeostasis. In the unicellular cyanobacterium, Synechocystis sp. PCC 6803, Fe and Mn deprivation resulted in distinct modifications of the physiological status. The effect on growth and photosynthetic activity under Fe limitation were more severe than those observed under Mn limitation. Moreover, the intracellular elemental quotas of Fe and Mn were found to be linked. Fe limitation reduced the intracellular Mn quota. Mn limitation did not exert a reciprocal effect on Fe quotas. Microarray analysis comparing Mn and Fe limitation revealed a stark difference in the extent of the transcriptional response to the two limiting conditions, reflective of the physiological responses. The effects of Fe limitation on the transcriptional network are widespread while the effects on Mn limitation are highly specific. Our analysis also revealed an overlap in the transcriptional response of specific Fe and Mn transporters. This overlap provides a framework for explaining Fe limitation induced changes in Mn quotas.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Homeostasis/genética , Hierro/metabolismo , Manganeso/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Proteínas de Transporte de Catión/genética , Immunoblotting , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , ARN no Traducido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Synechocystis/genética , Synechocystis/crecimiento & desarrollo , Synechocystis/metabolismoRESUMEN
Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence of circadian rhythms in Synechocystis.