RESUMEN

From the most ancient RNAs, which followed an RNY pattern and folded into small hairpins, modern RNA molecules evolved by two different pathways, dubbed Extended Genetic Code 1 and 2, finally conforming to the current standard genetic code. Herein, we describe the evolutionary path of the RNAome based on these evolutionary routes. In general, all the RNA molecules analysed contain portions encoded by both genetic codes, but crucial features seem to be better recovered by Extended 2 triplets. In particular, the whole Peptidyl Transferase Centre, anti-Shine-Dalgarno motif, and a characteristic quadruplet of the RNA moiety of RNAse-P are clearly unveiled. Differences between bacteria and archaea are also detected; in most cases, the biological sequences are more stable than their controls. We then describe an evolutionary trajectory of the RNAome formation, based on two complementary evolutionary routes: one leading to the formation of essentials, while the other complemented the molecules, with the cooperative assembly of their constituents giving rise to modern RNAs.

Asunto(s)

Archaea , Evolución Molecular , ARN , Archaea/genética , Bacterias/genética , Código Genético , Conformación de Ácido Nucleico , ARN/genética , ARN Bacteriano/genética

RESUMEN

Antimicrobial photodynamic therapy (aPDT) has shown efficacy in inactivating different bacterial species by photosensitizer-induced free radical production. Despite aPDT is considered unable to cause resistant strains, enzymatic pathways for detoxification of reactive oxygen species and transmembrane photosensitizer efflux systems could cause resistance to aPDT. Resistance mechanisms can be evaluated by measurement of mRNA from by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Thus, the aim of this study was to access the mRNA level data obtained by RT-qPCR in bacterial cells submitted to photodynamic therapy. Studies performed on mRNA levels in bacteria after PDT were assessed on MEDLINE/Pubmed. The mRNA levels from genes related to various functions have been successfully evaluated in both Gram-positive and -negative bacteria after aPDT by RT-qPCR. Such an approach has improved the understanding of aPDT-induced effects, and reinforced the effectiveness of aPDT on bacteria, which can cause infections in different human tissues.

Asunto(s)

Fotoquimioterapia , Fármacos Fotosensibilizantes , ARN Mensajero , Fotoquimioterapia/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Humanos , Bacterias/efectos de los fármacos , Bacterias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Bacteriano/análisis

RESUMEN

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

Asunto(s)

Brucella abortus , Células Endoteliales , Células Epiteliales , Antígenos de Histocompatibilidad Clase I , Interferón gamma , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , ARN Bacteriano/genética , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/metabolismo , Brucelosis/inmunología , Brucelosis/metabolismo , Brucelosis/microbiología , Brucelosis/genética , Aparato de Golgi/metabolismo , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Monocitos/metabolismo , Monocitos/inmunología , Monocitos/efectos de los fármacos

RESUMEN

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, m1A tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. M1A in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its m1A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified that acts on endogenous RNA modifications. Using Streptomyces venezuelae as a model organism, we confirmed the presence and quantitative m1A tRNA signatures using mass spectrometry and high-throughput tRNA sequencing. We identified two RNA demethylases that can remove m1A in tRNA and validated the activity of a previously annotated tRNA m1A writer. Using single-gene knockouts of these erasers and the m1A writer, we found dynamic changes of m1A levels in many tRNAs under stress conditions. Phenotypic characterization highlighted changes in their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms of translational regulation in bacteria.

Asunto(s)

Adenosina , ARN de Transferencia , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces/enzimología , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , ARN Bacteriano/metabolismo , Procesamiento Postranscripcional del ARN

RESUMEN

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

Asunto(s)

Brucella abortus , Regulación Bacteriana de la Expresión Génica , Brucella abortus/genética , Brucella abortus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Transcripción Genética , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , Estrés Fisiológico , Animales , Macrófagos/microbiología

RESUMEN

Cholesterol metabolism is important at the physiological level as well as in several diseases, with small RNA being an element to consider in terms of its epigenetic control. Thus, the aim of this study was to identify differences between bacterial small RNAs present at the gut level in hypercholesterolemic and normocholesterolemic individuals. Twenty stool samples were collected from hypercholesterolemic and normocholesterolemic subjects. RNA extraction and small RNA sequencing were performed, followed by bioinformatics analyses with BrumiR, Bowtie 2, BLASTn, DESeq2, and IntaRNA, after the filtering of the reads with fastp. In addition, the prediction of secondary structures was obtained with RNAfold WebServer. Most of the small RNAs were of bacterial origin and presented a greater number of readings in normocholesterolemic participants. The upregulation of small RNA ID 2909606 associated with Coprococcus eutactus (family Lachnospiraceae) was presented in hypercholesterolemic subjects. In addition, a positive correlation was established between small RNA ID 2149569 from the species Blautia wexlerae and hypercholesterolemic subjects. Other bacterial and archaeal small RNAs that interacted with the LDL receptor (LDLR) were identified. For these sequences, the prediction of secondary structures was also obtained. There were significant differences in bacterial small RNAs associated with cholesterol metabolism in hypercholesterolemic and normocholesterolemic participants.

Asunto(s)

Hipercolesterolemia , Humanos , Hipercolesterolemia/metabolismo , ARN Bacteriano/genética , Colesterol/metabolismo

RESUMEN

Bacterial small non-coding RNAs (sRNAs) play key roles as genetic regulators, mediating in the adaptability to changing environmental conditions and stress responses. In this work, we analysed putative sRNAs identified by RNA-seq experiments in different aeration conditions in the extremophile bacterium P. extremaustralis. These analyses allowed the identification of 177 putative sRNAs under aerobiosis (A), microaerobiosis (M) and microaerobiosis after H2 O2 exposure (m-OS). The size and transcription profile of eight sRNAs with differential expression were verified by Northern blot. sRNA40, with unknown function but conserved in other Pseudomonas species, was selected to perform overexpression experiments followed by RNA-seq analysis. The overexpression of sRNA40 in P. extremaustralis resulted in significant expression changes of 19 genes with 14 differentially upregulated and five downregulated. Among the upregulated genes, eight transcripts corresponded to components of secretion systems, such as gspH, gspK, and gspM, belonging to the Type II secretion system, and rspO and rspP from Type III secretion system. Our results showed a novel sRNA which expression was triggered by low oxygen levels, and whose overexpression was associated with upregulation of selected components of protein secretion systems.

Asunto(s)

Oxígeno , ARN Pequeño no Traducido , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo , Oxígeno/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética

RESUMEN

BACKGROUND: Salmonella Typhimurium is a Gram-negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella-containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin-encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post-transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post-transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S. Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild-type strain, suggesting that ompX mRNA is also regulated at a post-transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2-induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Porinas , Salmonella typhimurium , Animales , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Porinas/genética , Porinas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo

RESUMEN

Riboswitches are RNA sensors affecting post-transcriptional processes through their ability to bind to small molecules. Thiamine pyrophosphate (TPP) riboswitch plays a crucial role in regulating genes involved in synthesizing or transporting thiamine and phosphorylated derivatives in bacteria, archaea, plants, and fungi. Although TPP riboswitch is reasonably well known in bacteria, there is a gap in the knowledge of the fungal TPP riboswitches structure and dynamics, involving mainly sequence variation and TPP interaction with the aptamers. On the other hand, the increase of fungal infections and antifungal resistance raises the need for new antifungal therapies. In this work, we used computational approaches to build three-dimensional models for the three TPP riboswitches identified in Aspergillus oryzae, in which we studied their structure, dynamics, and binding free energy change (ΔGbind) with TPP. Interaction patterns between the TPP and the surrounding nucleotides were conserved among the three models, evidencing high structural conservation. Furthermore, we show that the TPP riboswitch from the A. oryzae NMT1 gene behaves similarly to the E. coli thiA gene concerning the ΔGbind. In contrast, mutations in the fungal TPP riboswitches from THI4 and the nucleoside transporter genes led to structural differences, affecting the binding-site volume, hydrogen bond occupancy, and ΔGbind. Besides, the number of water molecules surrounding TPP influenced the ΔGbind considerably. Notably, our ΔGbind estimation agreed with previous experimental data, reinforcing the relationship between sequence conservation and TPP interaction.

Asunto(s)

Aspergillus oryzae/genética , Biología Computacional , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Riboswitch , Escherichia coli/genética , Enlace de Hidrógeno , Conformación de Ácido Nucleico , ARN/química , ARN/genética , ARN Bacteriano , Relación Estructura-Actividad , Termodinámica

RESUMEN

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Asunto(s)

Animales , Ratones , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Porinas/genética , Porinas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología

RESUMEN

BACKGROUND: Aedes aegypti and Ae. albopictus are vectors of numerous arboviruses that adversely affect human health. In mosquito vectors of disease, the bacterial microbiota influence several physiological processes, including fertility and vector competence, making manipulation of the bacterial community a promising method to control mosquito vectors. In this study, we describe the reproductive tract tissue microbiota of lab-reared virgin Ae. aegypti and Ae. albopictus males, and virgin, mated, and mated + blood-fed females of each species, comparing the bacterial composition found there to the well-described gut microbiota. METHODS: We performed metabarcoding of the 16S rRNA isolated from the gut, upper reproductive tract (URT; testes or ovaries), and lower reproductive tract (LRT; males: seminal vesicles and accessory glands; females: oviduct, spermathecae, and bursa) for each species, and evaluated the influence of host species, tissue, nutritional status, and reproductive status on microbiota composition. Finally, based on the identified taxonomic profiles of the tissues assessed, bacterial metabolic pathway abundance was predicted. RESULTS: The community structure of the reproductive tract is unique compared to the gut. Asaia is the most prevalent OTU in the LRTs of both Ae. aegypti and Ae. albopictus. In the URT, we observed differences between species, with Wolbachia OTUs being dominant in the Ae. albopictus URT, while Enterobacter and Serratia were dominant in Ae. aegypti URT. Host species and tissue were the best predictors of the community composition compared to reproductive status (i.e., virgin or mated) and nutritional status (i.e., sugar or blood-fed). The predicted functional profile shows changes in the abundance of specific microbial pathways that are associated with mating and blood-feeding, like energy production in mated tissues and siderophore synthesis in blood-fed female tissues. CONCLUSIONS: Aedes aegypti and Ae. albopictus have distinct differences in the composition of microbiota found in the reproductive tract. The distribution of the bacterial taxonomic groups indicates that some bacteria have tissue-specific tropism for reproductive tract tissue, such as Asaia and Wolbachia. No significant differences in the taxonomic composition were observed in the reproductive tract between virgin, mated, and mated + blood-fed females, but changes in the abundance of specific metabolic pathways were found in the predicted microbial functional profiles in mated and blood-fed females.

Asunto(s)

Aedes/microbiología , Bacterias/clasificación , Microbiota , Mosquitos Vectores/parasitología , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Código de Barras del ADN Taxonómico , Femenino , Genitales/microbiología , Humanos , Especificidad de Órganos , ARN Bacteriano/genética , ARN Ribosómico 16S/genética

RESUMEN

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

Asunto(s)

Antiinfecciosos/química , Infecciones Bacterianas/tratamiento farmacológico , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Quinolonas/química , Antiinfecciosos/uso terapéutico , Infecciones Bacterianas/genética , Infecciones Bacterianas/microbiología , Girasa de ADN/efectos de los fármacos , Topoisomerasa de ADN IV/antagonistas & inhibidores , ADN Bacteriano/biosíntesis , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/patogenicidad , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/genética , Bacterias Grampositivas/patogenicidad , Humanos , Quinolonas/uso terapéutico , ARN Bacteriano/biosíntesis , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/uso terapéutico

RESUMEN

Background: In the absence of a late marker of treatment failure or relapse in MDR-TB patients, biomarkers based on host-miRNAs coupled with M. tuberculosis-RNAs evaluated in extracellular vesicles (EVs) are an alternative follow-up for MDR-TB disease. Characterization of EVs cargo to identify differentially expressed miRNAs before and after treatment, and to identify M. tuberculosis-derived RNA in serum EVs from resistant TB patients. Methods: EVs were isolated from serum of 26 drug-resistant TB (DR-TB) patients and 16 healthy subjects. Differential expression of miRNAs in pooled exosomes from both untreated and treated patients was assessed and individually validated at different time points during treatment. In addition, M. tuberculosis RNA was amplified in the same samples by qPCR. Results: A multivariate analysis using miR-let-7e-5p, -197-3p and -223-3p were found to be a more sensitive discriminator between healthy individuals and those with TB for both DR-TB (AUC= 0.96, 95%, CI=0.907-1) and MDR-TB groups (AUC= 0.95, 95%, CI= 0.89-1). Upregulation of miR-let-7e-5p were observed at the time of M. tuberculosis negative culture T(3-5) for MDR-TB group or for long-term T(9-15) for MDR-TB group without diabetes (T2DM). A second pathogen-based marker based on 30kDa and 5KST sequences was detected in 33% of the MDR-TB patients after the intensive phase of treatment. The miR-let7e-5p is a candidate biomarker for long-term monitoring of treatment for the group of MDR-TB without T2DM. A dual marker of host-derived miR-let7e-5p and M. tuberculosis-derived RNA for monitoring-TB treatment based in serum EVs. Conclusion: A dual marker consisting of host-derived miR-let7e-5p and M. tuberculosis-derived RNA, could be an indicator of treatment failure or relapse time after treatment was completed.

Asunto(s)

MicroARNs , Mycobacterium tuberculosis/genética , ARN Bacteriano/sangre , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Adulto , Anciano , Biomarcadores/sangre , Exosomas/genética , Exosomas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tuberculosis Resistente a Múltiples Medicamentos/sangre , Tuberculosis Resistente a Múltiples Medicamentos/genética , Adulto Joven

RESUMEN

In this study, we characterize the response of the free-living oligotrophic alphaproteobacterium Caulobacter crescentus to low temperatures by global transcriptomic analysis. Our results showed that 656 genes were upregulated and 619 were downregulated at least 2-fold after a temperature downshift. The identified differentially expressed genes (DEG) belong to several functional categories, notably inorganic ion transport and metabolism, and a subset of these genes had their expression confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Several genes belonging to the ferric uptake regulator (Fur) regulon were downregulated, indicating that iron homeostasis is relevant for adaptation to cold. Several upregulated genes encode proteins that interact with nucleic acids, particularly RNA: cspA, cspB, and the DEAD box RNA helicases rhlE, dbpA, and rhlB. Moreover, 31 small regulatory RNAs (sRNAs), including the cell cycle-regulated noncoding RNA (ncRNA) CcnA, were upregulated, indicating that posttranscriptional regulation is important for the cold stress response. Interestingly, several genes related to transport were upregulated under cold stress, including three AcrB-like cation/multidrug efflux pumps, the nitrate/nitrite transport system, and the potassium transport genes kdpFABC. Further characterization showed that kdpA is upregulated in a potassium-limited medium and at a low temperature in a SigT-independent way. kdpA mRNA is less stable in rho and rhlE mutant strains, but while the expression is positively regulated by RhlE, it is negatively regulated by Rho. A kdpA-deleted strain was generated, and its viability in response to osmotic, acidic, or cold stresses was determined. The implications of such variation in the gene expression for cold adaptation are discussed. IMPORTANCE 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. Here, we show that in Caulobacter crescentus, the expression of the genes involved in cation transport and homeostasis is altered in response to cold, which could lead to a decrease in iron uptake and an increase in nitrogen and high-affinity potassium transport by the Kdp system. This previously uncharacterized regulation of the Kdp transporter has revealed a new mechanism for adaptation to low temperatures that may be relevant for oligotrophic bacteria.

Asunto(s)

Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Proteínas Represoras/metabolismo , Proteínas Bacterianas/genética , Caulobacter crescentus/química , Caulobacter crescentus/genética , Frío , Transporte Iónico , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Regulón , Proteínas Represoras/genética

RESUMEN

BACKGROUND: 6S RNA is a regulator of cellular transcription that tunes the metabolism of cells. This small non-coding RNA is found in nearly all bacteria and among the most abundant transcripts. Lactic acid bacteria (LAB) constitute a group of microorganisms with strong biotechnological relevance, often exploited as starter cultures for industrial products through fermentation. Some strains are used as probiotics while others represent potential pathogens. Occasional reports of 6S RNA within this group already indicate striking metabolic implications. A conceivable idea is that LAB with 6S RNA defects may metabolize nutrients faster, as inferred from studies of Echerichia coli. This may accelerate fermentation processes with the potential to reduce production costs. Similarly, elevated levels of secondary metabolites might be produced. Evidence for this possibility comes from preliminary findings regarding the production of surfactin in Bacillus subtilis, which has functions similar to those of bacteriocins. The prerequisite for its potential biotechnological utility is a general characterization of 6S RNA in LAB. RESULTS: We provide a genomic annotation of 6S RNA throughout the Lactobacillales order. It laid the foundation for a bioinformatic characterization of common 6S RNA features. This covers secondary structures, synteny, phylogeny, and product RNA start sites. The canonical 6S RNA structure is formed by a central bulge flanked by helical arms and a template site for product RNA synthesis. 6S RNA exhibits strong syntenic conservation. It is usually flanked by the replication-associated recombination protein A and the universal stress protein A. A catabolite responsive element was identified in over a third of all 6S RNA genes. It is known to modulate gene expression based on the available carbon sources. The presence of antisense transcripts could not be verified as a general trait of LAB 6S RNAs. CONCLUSIONS: Despite a large number of species and the heterogeneity of LAB, the stress regulator 6S RNA is well-conserved both from a structural as well as a syntenic perspective. This is the first approach to describe 6S RNAs and short 6S RNA-derived transcripts beyond a single species, spanning a large taxonomic group covering multiple families. It yields universal insights into this regulator and complements the findings derived from other bacterial model organisms.

Asunto(s)

Regulación Bacteriana de la Expresión Génica , Lactobacillales/genética , ARN Bacteriano/genética , ARN no Traducido/genética , Bacillus subtilis/genética , Secuencia Conservada/genética , Humanos , Sintenía/genética

RESUMEN

The S. Typhi leuO gene, which codes for the LysR-type transcriptional regulator LeuO, contains five forward promoters named P3, P1, P2, P5 and P4, and two reverse promoters, P6 and P7. The activity of the forward promoters was revealed by primer extension using gene reporter fusions in an S. Typhi hns lrp mutant strain. Likewise, the activity of the reverse promoters was revealed in an hns background. Derepression of the transcription of the chromosomal gene was confirmed by RT-PCR in the hns lrp mutant. The leuOP1 transcriptional reporter fusion, which contained only the major P1 promoter, had a lower expression in a relA spoT mutant strain, indicating that the steady-state levels of the (p)ppGpp alarmone positively regulate it. In contrast, the leuOP3, leuOP5P4, leuOP6 and leuOP7 transcriptional fusions were derepressed in the relA spoT background, indicating that the alarmone has a negative effect on their expression. Thus, the search for genetic regulators and environmental cues that would differentially derepress leuO gene expression by antagonizing the action of the H-NS and Lrp nucleoid-associated proteins, or that would fine-tune the expression of the various promoters, will further our understanding of the significance that multiple promoters have in the control of LeuO expression.

Asunto(s)

Proteínas Bacterianas/genética , Regiones Promotoras Genéticas , Salmonella typhi/genética , Factores de Transcripción/genética , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Currently, there are increasing concerns about the possibility of a new epidemic due to emerging reports of Mayaro virus (MAYV) fever outbreaks in areas of South and Central America. Haemagogus mosquitoes, the primary sylvan vectors of MAYV are poorly characterized and a better understanding of the mosquito's viral transmission dynamics and interactions with MAYV and other microorganisms would be important in devising effective control strategies. In this study, a metatranscriptomic based approach was utilized to determine the prevalence of RNA viruses in field-caught mosquitoes morphologically identified as Haemagogus janthinomys from twelve (12) forest locations in Trinidad, West Indies. Known insect specific viruses including the Phasi Charoen-like and Humaiata-Tubiacanga virus dominated the virome of the mosquitoes throughout sampling locations while other viruses such as the avian leukosis virus, MAYV and several unclassified viruses had a narrower distribution. Additionally, assembled contigs from the Ecclesville location suggests the presence of a unique uncharacterized picorna-like virus. Mapping of RNA sequencing reads to reference mitochondrial sequences of potential feeding host animals showed hits against avian and rodent sequences, which putatively adds to the growing body of evidence of a potentially wide feeding host-range for the Haemagogus mosquito vector.

Asunto(s)

Culicidae/virología , Virus ARN/aislamiento & purificación , Viroma , Infecciones por Alphavirus/epidemiología , Infecciones por Alphavirus/virología , Animales , Secuencia de Bases , Aves , Culicidae/microbiología , Brotes de Enfermedades , Reservorios de Enfermedades/virología , Geografía Médica , Especificidad del Huésped , Insectos Vectores/virología , Filogenia , Proteobacteria/genética , Virus ARN/clasificación , Virus ARN/genética , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , Roedores , Togaviridae/genética , Togaviridae/aislamiento & purificación , Trinidad y Tobago/epidemiología , Viroma/genética

RESUMEN

A novel pathogenic strain Vibrio 20190611023 was isolated from the hepatopancreas of moribund cultured Penaeus vannamei suffering from black gill disease. This strain was identified as V. brasiliensis based on the phylogenetic analyses of 16S rDNA gene and five other housekeeping genes (i.e., gapA, ftsZ, mreB, topA and gyrB). Some biochemical features of this strain were determined with an API 20NE system, and its haemolytic activity was determined using a sheep blood agar plate. The pathogenicity of this isolate 20190611023 was confirmed by the experimental challenge tests and histopathological examinations. P. vannamei were challenged via reverse gavage with different doses of bacterial suspensions. The calculated median lethal dose (LD50 ) was (3.16 ± 1.78) × 105  CFU/g (body weight). Moreover, antibiotic susceptibility tests were performed, the results of which showed that the strain 20190611023 was sensitive to chloramphenicol, compound sulphamethoxazole, ciprofloxacin, doxycycline and oxacillin, but resistant to erythromycin, kanamycin, gentamicin, cefoperazone, ceftriaxone, cefamezin and piperacillin. To our knowledge, this is the first report for demonstrating V. brasiliensis as a shrimp pathogen, which expands the host range of V. brasiliensis infection. The present study highlights that more attention should be paid to this novel pathogen in intensive shrimp aquaculture.

Asunto(s)

Farmacorresistencia Bacteriana , Penaeidae/microbiología , Vibrio/clasificación , Animales , Antibacterianos/farmacología , Tipificación de Secuencias Multilocus , ARN Bacteriano/análisis , ARN Ribosómico 16S/análisis , Vibrio/efectos de los fármacos , Vibrio/genética

RESUMEN

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.

Asunto(s)

Escherichia coli/metabolismo , Glucosa/farmacología , Estabilidad del ARN/efectos de los fármacos , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Estrés Fisiológico/efectos de los fármacos , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Estabilidad del ARN/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Estrés Fisiológico/genética

RESUMEN

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Asunto(s)

Anaplasma/aislamiento & purificación , Anaplasmosis/epidemiología , Enfermedades de los Perros/epidemiología , Ehrlichia canis/aislamiento & purificación , Ehrlichiosis/veterinaria , Anaplasma/enzimología , Anaplasma/genética , Anaplasmosis/microbiología , Animales , Proteínas Bacterianas/análisis , Chaperonina 60/análisis , Citrato (si)-Sintasa/análisis , Enfermedades de los Perros/microbiología , Perros , Ehrlichia canis/enzimología , Ehrlichia canis/genética , Ehrlichiosis/epidemiología , Ehrlichiosis/microbiología , Grenada/epidemiología , Prevalencia , ARN Bacteriano/análisis , ARN Ribosómico 16S/análisis , ARN Ribosómico 23S/análisis