Widespread Protection of RNA Cleavage Sites by a Riboswitch Aptamer that Folds as a Compact Obstacle to Scanning by RNase E.

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here, we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5' end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.

Metabolism of Free Guanidine in Bacteria Is Regulated by a Widespread Riboswitch Class.

The guanidyl moiety is a component of fundamental metabolites, including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse, and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound, and several gene families that can alleviate guanidine toxicity exist.

Riboswitch control of bacterial RNA stability.

Although riboswitches have long been known to regulate translation initiation and transcription termination, a growing body of evidence indicates that they can also control bacterial RNA lifetimes by acting directly to hasten or impede RNA degradation. Ligand binding to the aptamer domain of a riboswitch can accelerate RNA decay by triggering a conformational change that exposes sites to endonucleolytic cleavage or by catalyzing the self-cleavage of a prefolded ribozyme. Alternatively, the conformational change induced by ligand binding can protect RNA from degradation by blocking access to an RNA terminus or internal region that would otherwise be susceptible to attack by an exonuclease or endonuclease. Such changes in RNA longevity often accompany a parallel effect of the same riboswitch on translation or transcription. Consequently, a single riboswitch aptamer may govern the function of multiple effector elements (expression platforms) that are co-resident within a transcript and act independently of one another.

Guanidinium export is the primal function of SMR family transporters.

The small multidrug resistance (SMR) family of membrane proteins is prominent because of its rare dual topology architecture, simplicity, and small size. Its best studied member, EmrE, is an important model system in several fields related to membrane protein biology, from evolution to mechanism. But despite decades of work on these multidrug transporters, the native function of the SMR family has remained a mystery, and many highly similar SMR homologs do not transport drugs at all. Here we establish that representative SMR proteins, selected from each of the major clades in the phylogeny, function as guanidinium ion exporters. Drug-exporting SMRs are all clustered in a single minority clade. Using membrane transport experiments, we show that these guanidinium exporters, which we term Gdx, are very selective for guanidinium and strictly and stoichiometrically couple its export with the import of two protons. These findings draw important mechanistic distinctions with the notably promiscuous and weakly coupled drug exporters like EmrE.

Riboswitch-Associated Guanidinium-Selective Efflux Pumps Frequently Transmitted on Proteobacterial Plasmids Increase Escherichia coli Biofilm Tolerance to Disinfectants.

Members of the small multidrug resistance (SMR) efflux pump family known as SugE (recently renamed Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistance plasmids in Gram-negative bacilli, but their functional role remains unclear, as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to (i) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, (ii) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similarly to chromosomally encoded Gdx members, and (iii) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in Escherichia coli strains BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence, annotated as "SugE(p)," predominated among the >140 plasmids we surveyed. Enterobacterales plasmids carrying sugE(p) possessed a guanidine II riboswitch similar to the upstream region of E. coligdx Cloning and expression of sugE(p), gdx, and emrE sequences into a low-copy-number expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure.IMPORTANCE This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid-encoded guanidinium-selective small multidrug resistance (Gdm/SugE) efflux pumps. These sequences are frequently monitored as biocide resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that enterobacterial gdm sequences transmitted on plasmids possess a guanidine II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (which possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC-containing compounds, the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern for clinical and food safety prevention measures.

Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring blaCMY-2 and blaSHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries.

The objective of this study was to elucidate the genetic and evolutionary relatedness of blaCMY-2- and blaSHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring blaCMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, blaSHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.

SugE belongs to the small multidrug resistance (SMR) protein family involved in tributyltin (TBT) biodegradation and bioremediation by alkaliphilic Stenotrophomonas chelatiphaga HS2.

Tributyltin (TBT) used in a variety of industrial processes, subsequent discharge into the environment, its fate, toxicity and human exposure are topics of current concern. TBT degradation by alkaliphilic bacteria may be a key factor in the remediation of TBT in high pH contaminated sites. In this study, Stenotrophomonas chelatiphaga HS2 were isolated and identified from TBT contaminated site in Mediterranean Sea. S. chelatiphaga HS2 has vigor capability to transform TBT into dibutyltin and monobutyltin (DBT and MBT) at pH 9 and 7% NaCl (w/v). A gene was amplified and characterized from strain HS2 as SugE protein belongs to SMR protein family, a reverse transcription polymerase chain reaction analysis confirmed that SugE protein involved in the TBT degradation by HS2 strain. TBT bioremediation was investigated in stimulated TBT contaminated sediment samples (pH 9) using S chelatiphaga HS2 in association with E. coli BL21 (DE3)-pET28a(+)-sugE instead of S chelatiphaga HS2 alone reduced significantly the TBT half-life from 12d to 5d, although no TBT degradation appeared using E. coli BL21 (DE3)-pET28a(+)-sugE alone. This finding indicated that SugE gene increased the rate and degraded amount of TBT and is necessary in enhancing TBT bioremediation.

Evaluación por gastroscopia simple y cromoendoscopia convencional de la superficie gastroesofágica y duodenal proximal del equino. Estudio piloto / Evaluation by simple gastroscopy and conventional chromoendoscopy of the gastro-esophageal and proximal duodenal surface of equine. Preliminary study

RESUMEN Las enfermedades gastrointestinales equinas tienen una alta incidencia con un pronóstico variable en la práctica clínica. La mayoría de los estudios se limitan a describir lesiones ulcerativas y lesiones inflamatorias. El objetivo de este estudio fue evaluar el potencial diagnóstico complementario de la cromoendoscopia convencional en la mucosa gas-troesofágica y duodenal proximal del equino. El estudio incluyó 20 caballos criollos colombianos de ambos sexos (12 hembras y 8 machos), con edades entre 5 y 20 años, peso entre 250 y 350 kilogramos, condición corporal 4-5/9 y con historial de alteraciones digestivas en los últimos 3 meses; quienes previo a la evaluación por gastroscopia y cromoendoscopia se sometieron a ayuno (sólidos 12h y líquidos 4h) y sedación (xilacina 0,5 mg/kg/iv). Se utilizaron tinciones como rojo fenol, lugol, índigo carmín, azul de metileno y ácido acético y se tomaron biopsias de los segmentos que mostraron reacción. El azul de metileno reveló 52% de las lesiones, el lugol 19%; por su parte, el rojo fenol, el índigo carmín y el ácido acético revelaron el 9,5% restante. El epitelio escamoso fue el más afectado (66,6%), el glandular (19%), antro pilórico (9,5%) y duodeno proximal (4,7%). Los hallazgos histopatológicos fueron hiperplasia, hipertrofia, hiperqueratosis, congestión, degeneración vacuolar, infiltrados celulares, fibrosis, necrosis y atrofia en diferentes grados de severidad. La cromoendoscopia reveló lesiones prematuras, que pasaron desapercibidas con las técnicas convencionales de endoscopia del tracto digestivo. Este es el primer estudio que emplea la cromoendoscopia en equinos; a pesar de que la técnica mejoró la visualización y facilitó la ubicación y descripción de lesiones ulcerativas prematuras a través de la histopatología, se recomiendan mayores estudios controlados y con un número más amplio de muestras.

ABSTRACT Equine gastrointestinal diseases have a high occurrence with a variable prognostic in clinic practice. Most of the studies limits to describe ulcerative and inflammatory lesions. The objective of this study was to evaluate the potential complementary diagnostic of conventional chromoendoscopy on the gastroesophageal and proximal duodenal mucosa of the equine. 20 Colombian creole horses, of both sexes (12 females and 8 males), with ages between 5 and 20 years old, weight between 250 and 350 kilograms, body condition 4-5/9, that had presented digestive alterations in the last 3 months, were subjected to fasting (solids 12h and liquids 4h) and sedated (xylazine 0,5 mg/kg/iv) to be evaluated by gastroscopy and chromoendoscopy, using for stains phenol red, lugol, indigo carmine, methylene blue and acetic acid, taking biopsy samples of the segments that showed reaction. The methylene blue revealed 52%, lugol 19%, and phenol red, indigo carmine and acetic acid revealed only 9,5% of the lesions, being the squamous epithelium the most affected (66,6%), glandular epithelium (19%), pyloric antrum (9,5%) and proximal duodenum (4,7%), where histopathological findings were hyperplasia, hypertrophy, hyperkeratosis, congestion, vacuolar degeneration, cellular infiltrates, fibrosis, necrosis and atrophy in different degrees of severity. Chromoendoscopy revealed lesions premature, which go unnoticed with conventional light endoscopy techniques. This is the first study using chromoscopy in horses to show that the reagents used allow a better visualization of injuries than the conventional technique, helping histopathological studies and molecular biology to understand ulcerative premature injuries and possible pathophysiological pathways. However, larger controlled studies and a larger number of samples are needed.

Cytosine DNA methylation influences drug resistance in Escherichia coli through increased sugE expression.

Escherichia coli K-12 strains contain the orphan cytosine-5 DNA methyltransferase enzyme Dcm (DNA cytosine methyltransferase). Two recent reports indicate that Dcm has an influence on stationary phase gene expression in E. coli. Herein, we demonstrate that dcm knockout cells overexpress the drug resistance transporter SugE, which has been linked to ethidium bromide (ETBR) resistance. SugE expression also increased in the presence of the DNA methylation inhibitor 5-azacytidine, suggesting that Dcm-mediated DNA methylation normally represses sugE expression. The effect of Dcm on sugE expression is primarily restricted to early stationary phase, and RpoS is required for robust sugE expression. Dcm knockout cells are more resistant to ETBR than wild-type cells, and complementation with a plasmid-borne dcm gene restores ETBR sensitivity. SugE knockout cells are more sensitive to ETBR than wild-type cells. These data indicate that Dcm influences the sensitivity to an antimicrobial compound through changes in gene expression.

Equine gastric ulcerative syndrome in Antioquia (Colombia): Frequency and risk factors / Síndrome ulcerativo gástrico equino en Antioquia (Colombia): Frecuencia y factores de riesgo / Síndrome da úlcera gástrica equina em Antioquia (Colombia): Frequência e fatores de risco

Abstract Background: Equine gastric ulcer syndrome (EGUS) has been associated with duration of confinement, grain supplementation, exercise, stress, use of non-steroidal anti-inflammatory drugs, and the gastric microbiome. On the other hand, limited information is available on the risk factors for EGUS in Colombia. Objective: To determine the frequency and risk factors for the presentation of EGUS in several municipalities of Antioquia. Methods: A total of 103 male and female horses were evaluated. Endoscopic samples from different portions of the stomach were taken and subjected to histopathological analysis. Information on management conditions for each animal was also obtained. Descriptive statistics, association analysis and logistic regression were performed. Results: A 69% of the animals presented at least one gastric lesion: 22.3% lesions in the squamous mucosa, 39.2% in the Margo plicatus (MP) and 48.9% in the glandular region. Diet was associated with the presentation of ulcers in the squamous mucosa (p = 0.003). MP ulceration was associated with concentrate feed consumption (p = 0.032). Conclusion: The EGUS frequency is 69%. Consumption of concentrate feed by horses in Antioquia is a critical factor for the development of ulcers in the stomach squamous and glandular regions.

Resumen Antecedentes: El síndrome ulcerativo gástrico equino (SUGE) se ha asociado a factores como estabulación, consumo de concentrado, ejercicio, estrés, uso de AINEs y microbiota del estómago. Por otro lado, la información sobre los factores de riesgo para EGUS en Colombia es limitada. Objetivo: Determinar la frecuencia y factores de riesgo para la presentación de SUGE en caballos de Medellín y municipios cercanos. Métodos: Fueron evaluados 103 equinos de ambos sexos. Se realizó análisis histopatológico a muestras tomadas por endoscopia en diferentes porciones del estómago. Adicionalmente, se obtuvo información sobre las condiciones de manejo de cada animal. Se realizó estadística descriptiva, análisis de asociación y regresión logística. Resultados: El 69% de los animales presentaron al menos una lesión gástrica, 22,3% lesiones en la mucosa escamosa, 39,2% en el Margo plicatus (MP) y 48,9% en la región glandular. La dieta estuvo asociada a la presentación de úlceras en la mucosa escamosa (p = 0,003). La ulceración en MP estuvo asociada al consumo de alimento concentrado (p = 0,032). Conclusión: La frecuencia de SUGE es del 69%. El consumo de alimento concentrado por equinos en Antioquia es un factor crítico para el desarrollo de úlceras en las regiones escamosa y glandular del estómago.

Resumo Antecedentes: A síndrome da úlcera gástrica equina (SUGE) tem sido associada a fatores como estabulação, subministro de ração, exercício, estrese, uso de AINE e microbiota do estômago. Por outro lado, a informação sobre os fatores de risco para o EGUS na Colômbia é limitada. Objetivo: Determinar a frequência e fatores de risco para a apresentação da SUGE em uma população de cavalos de Medellín e cidades próximas. Métodos: Foram estudados 103 equinos de ambos sexos. Amostras das diferentes porções do estômago tomadas pela endoscopia foram sometidas a análises histopatológico. Igualmente, dados das condições de manejo de cada animal foram obtidos. Se realizou estatística descritiva, análises de associação e regressão logística. Resultados: O 69% dos animais estudados apresentarão pelo menos uma lesão gástrica, 22,3% lesões na mucosa escamosa, 39,2% no Margo plicatus (MP) e 48,9% na região glandular. Se determinou que a dieta é um fator associado à apresentação de úlceras na mucosa escamosa (p = 0,003). A ulceração em MP foi associada ao subministro de ração (p = 0,032). Conclusão: A frequência da SUGE foi do 69%; a presença de ração na dieta é um fator crítico para o desenvolvimento de úlceras nas regiões escamosa e glandular.