RESUMEN
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
The D1 protein of photosystem II in the thylakoid membrane of photosynthetic organisms is encoded by psbA genes, which in cyanobacteria occur in the form of a small gene family. Light-dependent up-regulation of psbA gene expression is crucial to ensure the proper replacement of the D1 protein. To gain a high level of gene expression, psbA transcription can be enhanced by several orders of magnitude. Recent transcriptome analyses demonstrated a high number of cis-encoded antisense RNAs (asRNAs) in bacteria, but very little is known about their possible functions. Here, we show the presence of two cis-encoded asRNAs (PsbA2R and PsbA3R) of psbA2 and psbA3 from Synechocystis sp. PCC 6803. These asRNAs are located in the 5' untranslated region of psbA2 and psbA3 genes. Their expression becomes up-regulated by light and down-regulated by darkness, similar to their target mRNAs. In the PsbA2R-suppressing strain [PsbA2R(-)], the amount of psbA2 mRNA was only about 50% compared with the control strain. Likewise, we identified a 15% lowered activity of photosystem II and a reduced amount of the D1 protein in PsbA2R(-) compared with the control strain. The function of PsbA2R in the stabilization of psbA2 mRNA was shown from in vitro RNase E assay when the AU box and the ribosome-binding site in the 5' untranslated region of psbA2 mRNA were both covered by PsbA2R. These results add another layer of complexity to the mechanisms that contribute to psbA gene expression and show PsbA2R as a positively acting factor to achieve a maximum level of D1 synthesis.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Complejo de Proteína del Fotosistema II/metabolismo , ARN sin Sentido/genética , Synechocystis/metabolismo , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Oscuridad , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Luz , Datos de Secuencia Molecular , Mutagénesis , Fotosíntesis , Complejo de Proteína del Fotosistema II/genética , Estabilidad del ARN , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Synechocystis/genética , Synechocystis/crecimiento & desarrollo , Synechocystis/efectos de la radiación , Tilacoides/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg(2+), pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.
Asunto(s)
Endorribonucleasas/metabolismo , Prochlorococcus/enzimología , Prochlorococcus/genética , Estabilidad del ARN , ARN sin Sentido/metabolismo , ARN Mensajero/metabolismo , Bacteriófagos/fisiología , Endorribonucleasas/química , Endorribonucleasas/aislamiento & purificación , Genes de ARNr , Islas Genómicas , Prochlorococcus/virología , ARN Bicatenario/metabolismo , Transcripción GenéticaRESUMEN
Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in Gram-negative bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as Escherichia coli, feedback-regulate RNase E expression via the rne 5'-untranslated region (5' UTR) in cis. However, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. In this study, we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp. PCC 6803 after a brief exposure to UV. Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level. Moreover, the enzymatic activity of RNase E rapidly increased as well, although the protein stability decreased. This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination, but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation. These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.
RESUMEN
Previous studies have shown that infection of Prochlorococcus MED4 by the cyanophage P-SSP7 leads to increased transcript levels of host endoribonuclease (RNase) E. However, it has remained enigmatic whether this is part of a host defense mechanism to degrade phage messenger RNA (mRNA) or whether this single-strand RNA-specific RNase is utilized by the phage. Here we describe a hitherto unknown means through which this cyanophage increases expression of RNase E during phage infection and concomitantly protects its own RNA from degradation. We identified two functionally different RNase E mRNA variants, one of which is significantly induced during phage infection. This transcript lacks the 5' UTR, is considerably more stable than the other transcript, and is likely responsible for increased RNase E protein levels during infection. Furthermore, selective enrichment and in vivo analysis of double-stranded RNA (dsRNA) during infection revealed that phage antisense RNAs (asRNAs) sequester complementary mRNAs to form dsRNAs, such that the phage protein-coding transcriptome is nearly completely covered by asRNAs. In contrast, the host protein-coding transcriptome is only partially covered by asRNAs. These data suggest that P-SSP7 orchestrates degradation of host RNA by increasing RNase E expression while masking its own transcriptome from RNase E degradation in dsRNA complexes. We propose that this combination of strategies contributes significantly to phage progeny production.
Asunto(s)
Bacteriófagos/genética , Endorribonucleasas/genética , Prochlorococcus/genética , Transcriptoma/genética , Bacteriófagos/patogenicidad , Endorribonucleasas/biosíntesis , Genoma Viral , Interacciones Huésped-Patógeno/genética , Prochlorococcus/virología , Estabilidad del ARN/genética , ARN sin Sentido/biosíntesis , ARN sin Sentido/genética , ARN Bicatenario/genética , ARN Mensajero/genéticaRESUMEN
Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage.
Asunto(s)
Bacteriófagos/genética , Especificidad del Huésped , Metagenómica , Prochlorococcus/virología , Synechococcus/virología , Transcriptoma , Bacteriófagos/fisiología , Perfilación de la Expresión Génica , Océanos y Mares , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Prochlorococcus/genética , Alineación de Secuencia , Análisis de Secuencia de ARN , Synechococcus/genéticaRESUMEN
Regulatory RNAs play a central role in the regulation of gene expression and can act on several regulatory levels from transcriptional initiation and RNA processing to the control of initiation of translation and RNA stability. One class of these molecules is non-coding (nc)RNAs in bacteria that typically lack protein-coding potential, range in size between 50 and 500nt and originate from intergenic regions. Common methods for the identification of these RNAs are either based on computational predictions, or on transcriptomic analyses of laboratory cultures, whereas very little is known about ncRNAs in environmental microbial populations. Here, we have combined a metatranscriptomics approach with a selective enrichment protocol for ncRNAs. The primary objective of this study was the identification of novel, environmentally relevant ncRNAs focusing on the cyanobacterium Prochlorococcus, which was one of the dominant microorganisms of the marine community of the Gulf of Aqaba when samples were taken.