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1.
RNA Biol ; 14(11): 1544-1558, 2017 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-28296572

RESUMEN

Trans-encoded sRNA154 is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The sRNA154 deletion strain showed a significant decrease in growth under N-limitation, pointing toward a regulatory role of sRNA154 in N-metabolism. Aiming to elucidate its regulatory function we characterized sRNA154 by means of biochemical and genetic approaches. 24 homologs of sRNA154 were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loops. Transcriptome studies of sRNA154 deletion mutants by an RNA-seq approach uncovered nifH- and nrpA-mRNA, encoding the α-subunit of nitrogenase and the transcriptional activator of the nitrogen fixation (nif)-operon, as potential targets besides other components of the N-metabolism. Furthermore, results obtained from stability, complementation and western blot analysis, as well as in silico target predictions combined with electrophoretic mobility shift-assays, argue for a stabilizing effect of sRNA154 on the polycistronic nif-mRNA and nrpA-mRNA by binding with both loops. Further identified N-related targets were studied, which demonstrates that translation initiation of glnA2-mRNA, encoding glutamine synthetase2, appears to be affected by sRNA154 masking the ribosome binding site, whereas glnA1-mRNA appears to be stabilized by sRNA154. Overall, we propose that sRNA154 has a crucial regulatory role in N-metabolism in M. mazei by stabilizing the polycistronic mRNA encoding nitrogenase and glnA1-mRNA, as well as allowing a feed forward regulation of nif-gene expression by stabilizing nrpA-mRNA. Consequently, sRNA154 represents the first archaeal sRNA, for which a positive posttranscriptional regulation is demonstrated as well as inhibition of translation initiation.


Asunto(s)
Proteínas Arqueales/genética , Regulación de la Expresión Génica Arqueal , Genoma Arqueal , Methanosarcina/genética , Fijación del Nitrógeno/genética , ARN de Archaea/genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , Secuencia Conservada , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Methanosarcina/metabolismo , Nitrógeno/deficiencia , Conformación de Ácido Nucleico , Operón , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , ARN de Archaea/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN
2.
J Proteome Res ; 15(10): 3773-3783, 2016 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-27557128

RESUMEN

The recent discovery of an increasing number of small open reading frames (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. For biological and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. Consequently in the present study we evaluated analytical approaches that will allow for simultaneous analysis of widest parts of the proteome together with the predicted sORF. We performed a full proteome analysis of the methane producing archaeon Methanosarcina mazei strain Gö1 cytosolic proteome using a high/low pH reversed phase LC-MS bottom-up approach. The second analytical approach was based on semi-top-down strategy, encompassing a separation at intact protein level using a GelFree system, followed by digestion and LC-MS analysis. A high overlap in identified proteins was found for both approaches yielding the most comprehensive coverage of the cytosolic proteome of this organism achieved so far. The application of the second approach in combination with an adjustment of the search criteria for database searches further led to a significant increase of sORF peptide identifications, finally allowing to detect and identify 28 sORF gene products.


Asunto(s)
Methanosarcina/química , Péptidos/análisis , Proteoma/análisis , Cromatografía Liquida/métodos , Citosol/química , Minería de Datos , Espectrometría de Masas/métodos , Sistemas de Lectura Abierta/genética
3.
RNA Biol ; 11(5): 484-93, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24755959

RESUMEN

Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5'-region of two mRNAs in Methanosarcina mazei Gö1 and a few sRNAs that bind to 3'-UTRs in Sulfolobus solfataricus, three Pyrobaculum species, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5'-UTR or the 3'-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs.


Asunto(s)
Archaea/genética , ARN sin Sentido/genética , ARN de Archaea/genética , ARN Pequeño no Traducido/genética , Archaea/metabolismo , Emparejamiento Base , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica Arqueal , Genómica , ARN sin Sentido/metabolismo , ARN de Archaea/metabolismo , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética
4.
Biochem Soc Trans ; 41(1): 344-9, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23356309

RESUMEN

sRNAs (small non-coding RNAs) representing important players in many cellular and regulatory processes have been identified in all three domains of life. In Eukarya and Bacteria, functions have been assigned for many sRNAs, whereas the sRNA populations in Archaea are considerably less well characterized. Recent analyses on a genome-wide scale particularly using high-throughput sequencing techniques demonstrated the presence of high numbers of sRNA candidates in several archaea. However, elucidation of the molecular mechanism of sRNA action, as well as understanding their physiological roles, is in general still challenging, particularly in Archaea, since efficient genetic tools are missing. The identification of cellular targets of identified archaeal sRNAs by experimental approaches or computational prediction programs has begun only recently. At present, targets have been identified for one archaeal sRNA, sRNA162 in Methanosarcina mazei, which interacts with the 5' region of its targets, a cis-encoded and a trans-encoded target, blurring the paradigm of a border between cis- and trans-encoded sRNAs. Besides, the first experimental implications have been obtained in Haloarchaea and Pyrobaculum that archaeal sRNAs also target 3' regions of mRNAs. The present review summarizes our current knowledge on archaeal sRNAs and their biological functions and targets.


Asunto(s)
Archaea/genética , ARN de Archaea/genética , Procesamiento Postranscripcional del ARN
5.
Extremophiles ; 16(5): 685-96, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22763819

RESUMEN

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.


Asunto(s)
Archaea , Regulación de la Expresión Génica Arqueal/fisiología , Biosíntesis de Proteínas/fisiología , ARN de Archaea , ARN Pequeño no Traducido , Archaea/genética , Archaea/metabolismo , Secuencias Repetitivas Esparcidas
6.
Cell Metab ; 33(12): 2355-2366.e8, 2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34847376

RESUMEN

Hexokinases (HK) catalyze the first step of glycolysis limiting its pace. HK2 is highly expressed in gut epithelium, contributes to immune responses, and is upregulated during inflammation. We examined the microbial regulation of HK2 and its impact on inflammation using mice lacking HK2 in intestinal epithelial cells (Hk2ΔIEC). Hk2ΔIEC mice were less susceptible to acute colitis. Analyzing the epithelial transcriptome from Hk2ΔIEC mice during colitis and using HK2-deficient intestinal organoids and Caco-2 cells revealed reduced mitochondrial respiration and epithelial cell death in the absence of HK2. The microbiota strongly regulated HK2 expression and activity. The microbially derived short-chain fatty acid (SCFA) butyrate repressed HK2 expression via histone deacetylase 8 (HDAC8) and reduced mitochondrial respiration in wild-type but not in HK2-deficient Caco-2 cells. Butyrate supplementation protected wild-type but not Hk2ΔIEC mice from colitis. Our findings define a mechanism how butyrate promotes intestinal homeostasis and suggest targeted HK2-inhibition as therapeutic avenue for inflammation.


Asunto(s)
Colitis , Hexoquinasa , Animales , Células CACO-2 , Muerte Celular/fisiología , Colitis/metabolismo , Colitis/microbiología , Células Epiteliales/metabolismo , Hexoquinasa/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas Represoras/metabolismo
7.
Microbiologyopen ; 9(9): e1094, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32652897

RESUMEN

The associated microbiota of marine invertebrates plays an important role to the host in relation to fitness, health, and homeostasis. Cooperative and competitive interactions between bacteria, due to release of, for example, antibacterial substances and quorum sensing (QS)/quorum quenching (QQ) molecules, ultimately affect the establishment and dynamics of the associated microbial community. Aiming to address interspecies competition of cultivable microbes associated with emerging model species of the basal animal phyla Cnidaria (Aurelia aurita) and Ctenophora (Mnemiopsis leidyi), we performed a classical isolation approach. Overall, 84 bacteria were isolated from A. aurita medusae and polyps, 64 bacteria from M. leidyi, and 83 bacteria from ambient seawater, followed by taxonomically classification by 16S rRNA gene analysis. The results show that A. aurita and M. leidyi harbor a cultivable core microbiome consisting of typical marine ubiquitous bacteria also found in the ambient seawater. However, several bacteria were restricted to one host suggesting host-specific microbial community patterns. Interbacterial interactions were assessed by (a) a growth inhibition assay and (b) QS interference screening assay. Out of 231 isolates, 4 bacterial isolates inhibited growth of 17 isolates on agar plates. Moreover, 121 of the 231 isolates showed QS-interfering activities. They interfered with the acyl-homoserine lactone (AHL)-based communication, of which 21 showed simultaneous interference with autoinducer 2. Overall, this study provides insights into the cultivable part of the microbiota associated with two environmentally important marine non-model organisms and into interbacterial interactions, which are most likely considerably involved in shaping a healthy and resilient microbiota.


Asunto(s)
Bacterias/aislamiento & purificación , Ctenóforos/microbiología , Microbiota/fisiología , Escifozoos/microbiología , Acil-Butirolactonas/metabolismo , Animales , Bacterias/clasificación , Bacterias/crecimiento & desarrollo , Fenómenos Fisiológicos Bacterianos , Genes de ARNr , Interacciones Microbianas , Filogenia , Percepción de Quorum , ARN Ribosómico 16S/genética , Agua de Mar/microbiología
8.
J Innate Immun ; 11(5): 393-404, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30566939

RESUMEN

Animals are usually regarded as independent entities within their respective environments. However, within an organism, eukaryotes and prokaryotes interact dynamically to form the so-called metaorganism or holobiont, where each partner fulfils its versatile and crucial role. This review focuses on the interplay between microorganisms and multicellular eukaryotes in the context of host physiology, in particular aging and mucus-associated crosstalk. In addition to the interactions between bacteria and the host, we highlight the importance of viruses and nonmodel organisms. Moreover, we discuss current culturing and computational methodologies that allow a deeper understanding of underlying mechanisms controlling the physiology of metaorganisms.


Asunto(s)
Interacciones Microbiota-Huesped/fisiología , Microbiota/fisiología , Envejecimiento , Animales , Biología Computacional , Estado de Salud , Humanos , Modelos Biológicos , Moco/microbiología , Moco/virología , Simbiosis/fisiología
9.
Microbiol Spectr ; 6(4)2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-30027888

RESUMEN

Global (metabolic) regulatory networks allow microorganisms to survive periods of nitrogen starvation or general nutrient stress. Uptake and utilization of various nitrogen sources are thus commonly tightly regulated in Prokarya (Bacteria and Archaea) in response to available nitrogen sources. Those well-studied regulations occur mainly at the transcriptional and posttranslational level. Surprisingly, and in contrast to their involvement in most other stress responses, small RNAs (sRNAs) involved in the response to environmental nitrogen fluctuations are only rarely reported. In addition to sRNAs indirectly affecting nitrogen metabolism, only recently it was demonstrated that three sRNAs were directly involved in regulation of nitrogen metabolism in response to changes in available nitrogen sources. All three trans-acting sRNAs are under direct transcriptional control of global nitrogen regulators and affect expression of components of nitrogen metabolism (glutamine synthetase, nitrogenase, and PII-like proteins) by either masking the ribosome binding site and thus inhibiting translation initiation or stabilizing the respective target mRNAs. Most likely, there are many more sRNAs and other types of noncoding RNAs, e.g., riboswitches, involved in the regulation of nitrogen metabolism in Prokarya that remain to be uncovered. The present review summarizes the current knowledge on sRNAs involved in nitrogen metabolism and their biological functions and targets.


Asunto(s)
Nitrógeno/metabolismo , ARN de Archaea/metabolismo , ARN Bacteriano/metabolismo , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Sitios de Unión , Regulación de la Expresión Génica Arqueal , Regulación Bacteriana de la Expresión Génica , Glutamato-Amoníaco Ligasa/metabolismo , Nitrogenasa/metabolismo , ARN de Archaea/genética , ARN Bacteriano/genética , ARN Pequeño no Traducido , Transcriptoma
10.
Nat Microbiol ; 1(10): 16143, 2016 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-27670118

RESUMEN

Transcription termination sets the 3' end boundaries of RNAs and plays key roles in gene regulation. Although termination has been well studied in bacteria, the signals that mediate termination in archaea remain poorly understood. Here, we applied term-seq to comprehensively map RNA 3' termini, with single-base precision, in two phylogenetically distant archaea: Methanosarcina mazei and Sulfolobus acidocaldarius. Comparison of RNA 3' ends across hundreds of genes revealed the sequence composition of transcriptional terminators in each organism, highlighting both common and divergent characteristics between the different archaeal phyla. We find that, in contrast to bacteria, a considerable portion of archaeal genes are controlled by multiple consecutive terminators, generating several alternative 3' untranslated region isoforms for >30% of the genes. These alternative isoforms often present marked length differences, implying that archaea can employ regulation via alternative 3' untranslated regions, similar to eukaryotes. Although most of the terminators are intergenic, we discover numerous cases in which termination of one gene occurs within the coding region of a downstream gene, implying that leaky termination may tune inter-transcript stoichiometry in multi-gene operons. These results provide the first high-throughput maps of transcriptional terminators in archaea and point to an evolutionary path linking bacterial and eukaryal non-coding regulatory strategies.


Asunto(s)
Regiones no Traducidas 3' , Archaea/genética , Regulación de la Expresión Génica Arqueal , Isoformas de ARN/genética , ARN de Archaea/genética , Terminación de la Transcripción Genética , Proteínas Arqueales/genética , Perfilación de la Expresión Génica , Genes Arqueales , ARN no Traducido
11.
Biochimie ; 117: 138-48, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25890157

RESUMEN

In Methanosarcina mazei several small RNAs have been identified containing a small putative open reading frame (sORF) and thus classified as spRNAs. Here, we report on the first detection of three small proteins in M. mazei encoded by spRNAs using LC-MS/MS analysis of total protein extracts of cells grown under various stress conditions. Each spRNA shows high conservation in Methanosarcina species with regard to the sORF and the flanking non-coding RNA regions, moreover the predicted RNA structures are as well highly conserved. Characterizing the respective transcript levels in response to several stress conditions by northern blots demonstrated an enormous decrease of spRNA36 and spRNA44 during stationary growth (to less than 5%), and a significant increase of spRNA36 (2.5-fold) in response to nitrogen limitation. spRNA41, however, was only detected by RNA-Seq approaches. Quantification of the small proteins by LC-MS/MS using synthetic stable isotope labeled oligopeptides as standards indicated that the concentration of oligopetide36 and 41 in mid exponential phase is induced under nitrogen limitation, which in case of oligopeptide36 is in accordance with its transcript level. The relative amount of the three oligopeptides did not change upon entering stationary growth phase, even though the transcript levels decreased dramatically. Additional production of the oligopeptides in M. mazei did not result in any evident phenotype under standard or nitrogen limiting growth conditions. However, overall the transcript levels of several genes involved in carbon metabolism or in heat shock response were reduced 2-3 fold due to the overproduction, though no sORF specific change was observed. Based on our findings we hypothesize that oligopeptide36 might have a regulatory function in nitrogen metabolism by modulating the activity of a yet unknown target protein involved in the central nitrogen metabolism.


Asunto(s)
Proteínas Arqueales/genética , Methanosarcina/genética , Sistemas de Lectura Abierta/genética , ARN de Archaea/genética , Secuencia de Aminoácidos , Anaerobiosis , Proteínas Arqueales/metabolismo , Northern Blotting , Cromatografía Liquida , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica Arqueal/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genoma Arqueal/genética , Methanosarcina/crecimiento & desarrollo , Methanosarcina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligopéptidos/genética , Oligopéptidos/metabolismo , ARN de Archaea/química , ARN de Archaea/metabolismo , ARN no Traducido/genética , Cloruro de Sodio/farmacología , Espectrometría de Masas en Tándem , Temperatura
12.
Front Microbiol ; 5: 353, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25071757

RESUMEN

Although in nature most microorganisms are known to occur predominantly in consortia or biofilms, data on archaeal biofilm formation are in general scarce. Here, the ability of three methanoarchaeal strains, Methanobrevibacter smithii and Methanosphaera stadtmanae, which form part of the human gut microbiota, and the Methanosarcina mazei strain Gö1 to grow on different surfaces and form biofilms was investigated. All three strains adhered to the substrate mica and grew predominantly as bilayers on its surface as demonstrated by confocal laser scanning microscopy analyses, though the formation of multi-layered biofilms of Methanosphaera stadtmanae and Methanobrevibacter smithii was observed as well. Stable biofilm formation was further confirmed by scanning electron microscopy analysis. Methanosarcina mazei and Methanobrevibacter smithii also formed multi-layered biofilms in uncoated plastic µ-dishes(TM), which were very similar in morphology and reached a height of up to 40 µm. In contrast, biofilms formed by Methanosphaera stadtmanae reached only a height of 2 µm. Staining with the two lectins ConA and IB4 indicated that all three strains produced relatively low amounts of extracellular polysaccharides most likely containing glucose, mannose, and galactose. Taken together, this study provides the first evidence that methanoarchaea can develop and form biofilms on different substrates and thus, will contribute to our knowledge on the appearance and physiological role of Methanobrevibacter smithii and Methanosphaera stadtmanae in the human intestine.

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