RESUMEN
Translation, the decoding of mRNA into protein, is the third and final element of the central dogma. The ribosome, a nucleoprotein particle, is responsible and essential for this process. The bacterial ribosome consists of three rRNA molecules and approximately 55 proteins, components that are put together in an intricate and tightly regulated way. When finally matured, the quality of the particle, as well as the amount of active ribosomes, must be checked. The focus of this review is ribosome biogenesis in Escherichia coli and its cross-talk with the ongoing protein synthesis. We discuss how the ribosomal components are produced and how their synthesis is regulated according to growth rate and the nutritional contents of the medium. We also present the many accessory factors important for the correct assembly process, the list of which has grown substantially during the last few years, even though the precise mechanisms and roles of most of the proteins are not understood.
Asunto(s)
Escherichia coli/genética , Biosíntesis de Proteínas/fisiología , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Modelos Genéticos , Operón , Estructura Terciaria de Proteína , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
BACKGROUND: Mucosal infections elicit inflammatory responses via regulated signaling pathways. Infection outcome depends strongly on early events occurring immediately when bacteria start interacting with cells in the mucosal membrane. Hitherto reported transcription profiles on host-pathogen interactions are strongly biased towards in vitro studies. To detail the local in vivo genetic response to infection, we here profiled host gene expression in a recent experimental model that assures high spatial and temporal control of uropathogenic Escherichia coli (UPEC) infection within the kidney of a live rat. RESULTS: Transcriptional profiling of tissue biopsies from UPEC-infected kidney tissue revealed 59 differentially expressed genes 8 h post-infection. Their relevance for the infection process was supported by a Gene Ontology (GO) analysis. Early differential expression at 3 h and 5 h post-infection was of low statistical significance, which correlated to the low degree of infection. Comparative transcriptomics analysis of the 8 h data set and online available studies of early local infection and inflammation defined a core of 80 genes constituting a "General tissue response to early local bacterial infections". Among these, 25% were annotated as interferon-γ (IFN-γ) regulated. Subsequent experimental analyses confirmed a systemic increase of IFN-γ in rats with an ongoing local kidney infection, correlating to splenic, rather than renal Ifng induction and suggested this inter-organ communication to be mediated by interleukin (IL)-23. The use of comparative transcriptomics allowed expansion of the statistical data handling, whereby relevant data could also be extracted from the 5 h data set. Out of the 31 differentially expressed core genes, some represented specific 5 h responses, illustrating the value of comparative transcriptomics when studying the dynamic nature of gene regulation in response to infections. CONCLUSION: Our hypothesis-free approach identified components of infection-associated multi-cellular tissue responses and demonstrated how a comparative analysis allows retrieval of relevant information from lower-quality data sets. The data further define marked representation of IFN-γ responsive genes and a prompt inter-organ communication as a hallmark of an early local tissue response to infection.
Asunto(s)
Infecciones por Escherichia coli/genética , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Enfermedades Renales/genética , Riñón/metabolismo , Animales , Análisis por Conglomerados , Biología Computacional , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/microbiología , Interferón gamma/metabolismo , Riñón/microbiología , Riñón/patología , Enfermedades Renales/microbiología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Bazo/metabolismo , Escherichia coli Uropatógena/patogenicidadRESUMEN
A bacterial infection is accompanied by dynamic alterations in tissue homeostasis within the infected organ. What starts as a local bacterium-host cell interaction at the site of infection changes over time to include distant signaling and the engagement of multiple cell types in an effort to eradicate the bacteria. Recent advancements in imaging technologies, such as multiphoton microscopy, provide new tools to visualize the realtime dynamics of infection within the living host. The use of live animal models means that all of the interplaying factors, such as the immune, lymphatic, nervous, and vascular systems, are present and can be accounted for. This review describes novel insights of innate immune defense mechanisms obtained using real-time visualization of the infected tissue in a live animal model. This emerging field of "tissue microbiology" will provide data that, when combined with the massive knowledge base generated from research in "cellular microbiology," will provide a more complete picture of the complex infection process.
Asunto(s)
Infecciones Bacterianas/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/inmunología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Infecciones Bacterianas/microbiologíaRESUMEN
In Escherichia coli, release factor 1 (RF1) is one of two RFs that mediate termination; it specifically recognizes UAA and UAG stop codons. A mutant allele, prfA1, coding for an RF1 that causes temperature-sensitive (Ts) growth at 42 degrees C, was used to select for temperature-resistant (Ts(+)) suppressors. This study describes one such suppressor that is the result of an IS10 insertion into the cysB gene, giving a Cys(-) phenotype. CysB is a transcription factor regulating the cys regulon, mainly as an activator, which explains the Cys(-) phenotype. We have found that suppression is a consequence of the lost ability to donate sulfur to enzymes involved in the synthesis of thiolated nucleosides. From genetic analyses we conclude that it is the lack of the 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) modification of the wobble base of tRNA(Glu), tRNA(Lys), and/or tRNA(Gln) that causes the suppressor phenotype.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Factores de Terminación de Péptidos/genética , ARN de Transferencia de Glutamina , ARN de Transferencia de Ácido Glutámico , ARN de Transferencia de Lisina , Supresión Genética , Temperatura , Alelos , Proteínas Bacterianas/metabolismo , Codón de Terminación , Medios de Cultivo , Elementos Transponibles de ADN , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Calor , Factores de Terminación de Péptidos/metabolismo , Fenotipo , ARN de Transferencia de Glutamina/química , ARN de Transferencia de Glutamina/genética , ARN de Transferencia de Glutamina/metabolismo , ARN de Transferencia de Ácido Glutámico/química , ARN de Transferencia de Ácido Glutámico/genética , ARN de Transferencia de Ácido Glutámico/metabolismo , ARN de Transferencia de Lisina/química , ARN de Transferencia de Lisina/genética , ARN de Transferencia de Lisina/metabolismo , Tiouridina/análogos & derivados , Tiouridina/metabolismoRESUMEN
Release factor one (RF1) terminates protein synthesis in response to stop codons UAG and UAA. A mutant allele of RF1 causes temperature sensitive growth at 42 degrees C. We have earlier described the isolation of a suppressor of the temperature sensitive phenotype. The suppressor mutation is a small deletion in the open reading frame yrdC, and we have shown that the DeltayrdC mutation leads to immature 30S subunits and, as a consequence, to fewer translating ribosomes. YrdC is a small conserved protein with a dsRNA-binding surface. Here, we have characterized the YrdC protein. We show that the deletion leads to no production of functional protein, and we have indications that the YrdC protein might be essential in a wild type background. The protein is needed for the maturation of 16S rRNA, even though it does not interact tightly with either of the ribosomal subunits, or the 70S particles. The less effective maturation of rRNA affects the ribosomal feedback control, leading to an increase in expression from P1rrnB. We suggest that the function of the YrdC protein is to keep an rRNA structure needed for proper processing of 16S rRNA, especially at lower temperatures. This activity may require other factor(s). We suggest the gene be renamed rimN, and the mutant allele rimN141.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Unión al ARN/genética , Codón de Terminación , Cartilla de ADN , Genotipo , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Bicatenario/genética , ARN Ribosómico 16S/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/genética , Eliminación de SecuenciaRESUMEN
OBJECTIVES: Rituximab (RTX) is a biological treatment used off-label in patients with systemic lupus erythematosus (SLE). This survey aimed to investigate the off-label use of RTX in Europe and compare the characteristics of patients receiving RTX with those receiving conventional therapy. METHODS: Data on patients with SLE receiving RTX were taken from the International Registry for Biologics in SLE retrospective registry and complemented with data on patients with SLE treated with conventional therapy. For nationwide estimates of RTX use in patients with SLE, investigators were asked to provide data through case report forms (CRFs). Countries for which no data were submitted through CRFs, published literature and/or personal communication were used, and for European countries where no data were available, estimates were made on the assumption of similarities with neighbouring countries. RESULTS: The estimated off-label use of RTX in Europe was 0.5%-1.5% of all patients with SLE. In comparison with patients with SLE on conventional therapy, patients treated with RTX had longer disease duration, higher disease activity and were more often treated with immunosuppressives. The most frequent organ manifestations for which either RTX or conventional therapy was initiated were lupus nephritis followed by musculoskeletal and haematological. The reason for treatment was, besides disease control, corticosteroid-sparing for patients treated with conventional therapy. CONCLUSIONS: RTX use for SLE in Europe is restrictive and appears to be used as a last resort in patients for whom other reasonable options have been exhausted.
RESUMEN
Readthrough of the nonsense codons UAG, UAA, and UGA is seen in Escherichia coli strains lacking tRNA suppressors. Earlier results indicate that UGA is miscoded by tRNA(Trp). It has also been shown that tRNA(Tyr) and tRNA(Gln) are involved in UAG and UAA decoding in several eukaryotic viruses as well as in yeast. Here we have investigated which amino acid(s) is inserted in response to the nonsense codons UAG and UAA in E. coli. To do this, the stop codon in question was introduced into the staphylococcal protein A gene. Protein A binds to IgG, which facilitates purification of the readthrough product. We have shown that the stop codons UAG and UAA direct insertion of glutamine, indicating that tRNA(Gln) can read the two codons. We have also confirmed that tryptophan is inserted in response to UGA, suggesting that it is read by tRNA(Trp).
Asunto(s)
Codón sin Sentido , Escherichia coli/genética , Glutamina/genética , Biosíntesis de Proteínas , ARN de Transferencia de Glutamina/genética , ARN de Transferencia de Triptófano/genética , Análisis de Secuencia de ADN , Supresión GenéticaRESUMEN
BACKGROUND: Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD+ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD+, especially for one of the alleles, nadD72. RESULTS: In this study these two mutant proteins have been further characterized together with ten new mutant variants. Of the, in total, twelve mutations four are in a conserved motif in the C-terminus and eight are in the active site. We have tested the activity of the enzymes in vitro and their effect on the growth phenotype in vivo. There is a very good correlation between the two data sets. CONCLUSION: The mutations in the C-terminus did not reveal any function for the conserved motif. On the other hand, our data has lead us to assign amino acid residues His-19, Arg-46 and Asp-109 to the active site. We have also shown that the nadD gene is essential for growth in E. coli.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Mutación , Nicotinamida-Nucleótido Adenililtransferasa/química , Nicotinamida-Nucleótido Adenililtransferasa/genética , Sitios de Unión/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Conformación ProteicaRESUMEN
Together with release factor (RF) 2, RF1 recognises the stop codons and triggers the hydrolysis of the nascent peptide from peptidyl-tRNA during translation termination. prfA, the gene that codes for RF1, is located at 27 min on the Escherichia coli map as the second gene in the hemA-operon. The concentration of RF1 has been shown to increase with increased growth rate, but it is not known where and how this control is exerted. In this study we show that the growth rate regulation of RF1, at least in part, is controlled at P(hemA1), one of two promoters preceding the hemA gene. We have also characterised two mutations, asuA1 and asuA2, that are antisuppressors to the tRNA suppressor Su2. Our data indicate that the antisuppressor phenotype is caused by an increased amount of RF1. The asuA2 mutation is a G to an A change just downstream of the -10 region of P(hemA1), it leads to a higher concentration of RF1 in the cell and abolishes the growth rate regulation. This indicates that the sequence between the -10 region and the transcription start site is important for growth rate control. The increase in concentration of RF1 caused by asuA1 is most likely at the translational level. The efficiency of translation initiation of prfA is low due to a long distance between the start codon and the Shine-Dalgarno (SD) sequence. The asuA1 mutation creates a new start codon with a more optimal distance to the SD sequence. This leads to an increased expression of RF1, probably due to increased initiation efficiency.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Terminación de Péptidos/genética , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the rplM-rpsI operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the yrdC open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1.
Asunto(s)
Mutación , Factores de Terminación de Péptidos/fisiología , ARN Ribosómico 16S/fisiología , Temperatura , Secuencia de Bases , Datos de Secuencia Molecular , Operón , ARN Ribosómico 23S/fisiología , Ribosomas/fisiologíaRESUMEN
Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 degrees C but the level is still sufficient to support normal growth. At 42 degrees C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 degrees C, while a large decrease leads to a more pleiotropic phenotype.