The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License.Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB(V) superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn(2+) or Mg(2+) cations in a dosage-dependent manner. However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+). Furthermore, MobMN199 exhibited a high affinity binding for Mn(2+) but not for Mg(2+). We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.Funding for open access charge: Spanish Ministry of Science and Innovation [grants CSD2008-00013, INTERMODS to M.E.; BFU2008-02372/BMC, PRODNA to M.C.; BFU2009-10052 and CIBERES (an initiative of the Carlos III Spanish Health Institute) to M.M.]; European Union (grant EU-CP223111, CAREPNEUMO to M.E.); National Institutes of Health (grant GM61017 to J.F.S.); The Carlos III Spanish Health Institute, fellowship BF03/00529 (to F.L.-D.).Peer Reviewe

Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

FUNDING This study was financially supported by grants BIO2016-76412- C2-2-R (AEI/FEDER, UE) to AB from the Spanish Ministry of Economy and Competitiveness, and PID2019-104553RB-C21 to AB from the Spanish Ministry of Science and Innovation. ACKNOWLEDGMENTS Thanks are due to F. W. Studier for his gift of the E. coli BL21 (DE3) strain and to L. Rodríguez for her technical help in protein purification.Peer reviewedPublisher PD

Variability of the Pr77 sequence of L1Tc retrotransposon among six T. cruzi strains belonging to different discrete typing units (DTUs)

All trypanosomatid genomes are colonized by non-LTR retrotransposons which exhibit a highly conserved 77-nt sequence at their 5′ ends, known as the Pr77-hallmark (Pr77). The wide distribution of Pr77 is expected to be related to the gene regulation processes in these organisms as it has promoter and HDV-like ribozyme activities at the DNA and RNA levels, respectively. The identification of Pr77 hallmark-bearing retrotransposons and the study of the associations of mobile elements with relevant genes have been analyzed in the genomes of six strains of Trypanosoma cruzi belonging to different discrete typing units (DTUs) and with different geographical origins and host/vectors. The genomes have been sequenced, assembled and annotated. BUSCO analyses indicated a good quality for the assemblies that were used in comparative analyses. The results show differences among the six genomes in the copy number of genes related to virulence processes, the abundance of retrotransposons bearing the Pr77 sequence and the presence of the Pr77 hallmarks not associated with retroelements. The analyses also show frequent associations of Pr77-bearing retrotransposons and single Pr77 hallmarks with genes coding for trans-sialidases, RHS, MASP or hypothetical proteins, showing variable proportion depending on the type of retroelement, gene class and parasite strain. These differences in the genomic distribution of active retroelements and other Pr77-containing elements have shaped the genome architecture of these six strains and might be contributing to the phenotypic variability existing among the

11β-HSD2 SUMOylation Modulates Cortisol-induced Mineralocorticoid Receptor Nuclear Translocation Independently of Effects on Transactivation

The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) has an essential role in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor (MR) by converting 11β-hydroxyglucocorticoids to inactive 11-ketosteroids. Congenital deficiency of 11β-HSD2 causes a form of salt-sensitive hypertension known as the syndrome of apparent mineralocorticoid excess. The disease phenotype, which ranges from mild to severe, correlates well with reduction in enzyme activity. Furthermore, polymorphisms in the 11β-HSD2 coding gene (HSD11B2) have been linked to high blood pressure and salt sensitivity, major cardiovascular risk factors. 11β-HSD2 expression is controlled by different factors such as cytokines, sex steroids, or vasopressin, but posttranslational modulation of its activity has not been explored. Analysis of 11β-HSD2 sequence revealed a consensus site for conjugation of small ubiquitin-related modifier (SUMO) peptide, a major posttranslational regulatory event in several cellular processes. Our results demonstrate that 11β-HSD2 is SUMOylated at lysine 266. Non-SUMOylatable mutant K266R showed slightly higher substrate affinity and decreased Vmax, but no effects on protein stability or subcellular localization. Despite mild changes in enzyme activity, mutant K266R was unable to prevent cortisol-dependent MR nuclear translocation. The same effect was achieved by coexpression of wild-type 11β-HSD2 with sentrin-specific protease 1, a protease that catalyzes SUMO deconjugation. In the presence of 11β-HSD2-K266R, increased nuclear MR localization did not correlate with increased response to cortisol or increased recruitment of transcriptional coregulators. Taken together, our data suggests that SUMOylation of 11β-HSD2 at residue K266 modulates cortisol-mediated MR nuclear translocation independently of effects on transactivation

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria

The O3N2 and N2 abundance indicators revisited: improved calibrations based on CALIFA and T e-based literature data

Astronomy and Astrophysics 559 (2013): A114 reproduced with permission from Astronomy and AstrophysicsThe use of integral field spectroscopy is since recently allowing to measure the emission line fluxes of an increasingly large number of star-forming galaxies, both locally and at high redshift. Many studies have used these fluxes to derive the gas-phase metallicity of the galaxies by applying the so-called strong-line methods. However, the metallicity indicators that these datasets use were empirically calibrated using few direct abundance data points (Te-based measurements). Furthermore, a precise determination of the prediction intervals of these indicators is commonly lacking in these calibrations. Such limitations might lead to systematic errors in determining the gas-phase metallicity, especially at high redshift, which might have a strong impact on our understanding of the chemical evolution of the Universe. The main goal of this study is to review the most widely used empirical oxygen calibrations, O3N2 and N2, by using newdirect abundance measurements. We pay special attention to (1) the expected uncertainty of these calibrations as a function of the index value or abundance derived and (2) the presence of possible systematic offsets. This is possible thanks to the analysis of the most ambitious compilation of Te-based H ii regions to date. This new dataset compiles the Te-based abundances of 603 H ii regions extracted from the literature but also includes new measurements from the CALIFA survey. Besides providing new and improved empirical calibrations for the gas abundance, we also present a comparison between our revisited calibrations with a total of 3423 additional CALIFA H ii complexes with abundances derived using the ONS calibration from the literature. The combined analysis of T e-based and ONS abundances allows us to derive their most accurate calibration to date for both the O3N2 and N2 single-ratio indicators, in terms of all statistical significance, quality, and coverage of the parameters space. In particular, we infer that these indicators show shallower abundance dependencies and statistically significant offsets compared to others'. The O3N2 and N2 indicators can be empirically applied to derive oxygen abundances calibrations from either direct abundance determinations with random errors of 0.18 and 0.16, respectively, or from indirect ones (but based on a large amount of data), reaching an average precision of 0.08 and 0.09 dex (random) and 0.02 and 0.08 dex (systematic; compared to the direct estimations), respectivelyR.A. Marino is funded by the Spanish program of International Campus of Excellence Moncloa (CEI). D. Mast thank the Plan Nacional de Investigación y Desarrollo funding programs, AYA2012-31935 of the Spanish Ministerio de Economía y Competitividad, for the support given to this project. S.F.S thanks the the Ramón y Cajal project RyC-2011-07590 of the spanish Ministerio de Economía y Competitividad, for the support giving to this project. F.F.R.O. acknowledges the Mexican National Council for Science and Technology (CONACYT) for financial support under the program Estancias Postdoctorales y Sabáticas al Extranjero para la Consolidación de Grupos de Investigación, 2010-2012. We acknowledge financial support for the ESTALLIDOS collaboration by the Spanish Ministerio de Ciencia e Innovación under grant AYA2010- 21887-C04-03. BG-L also acknowledges support from the Spanish Ministerio de Economía y Competitividad (MINECO) under grant AYA2012- 39408-C02-02. J.F.-B. acknowledges financial support from the Ramón y Cajal Program and grant AYA2010-21322-C03-02 from the Spanish Ministry of Economy and Competitiveness (MINECO), as well as to the DAGAL network from the People’s Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013/ under REA grant agreement number PITN-GA-2011-289313. CK has been funded by project AYA2010-21887 from the Spanish PNAYA. P.P. acknowledges support by the Fundação para a Ciência e a Tecnologia (FCT) under project FCOMP-01-0124-FEDER-029170 (Reference FCT PTDC/FIS-AST/3214/2012), funded by FCT-MEC (PIDDAC) and FEDER (COMPETE). R.M.G.D. and R.G.B. also acknowledge support from the Spanish Ministerio de Economía y Competitividad (MINECO) under grant AyA2010-15081. V.S., L.G., and A.M.M. acknowledge financial support from the Fundação para a Ciência e a Tecnologia (FCT) under program Ciência 2008 and the research grant PTDC/CTE-AST/112582/200

The upper-airway microbiome as a biomarker of asthma exacerbations despite inhaled corticosteroid treatment.

BACKGROUND: The response to inhaled corticosteroids (ICS) in asthma is affected by the interplay of several factors. Among these, the role of the upper-airway microbiome has been scarcely investigated. We aimed to evaluate the association between the salivary, pharyngeal, and nasal microbiome with asthma exacerbations despite receipt of ICS. METHODS: Samples from 250 asthma patients from the Genomics and Metagenomics of Asthma Severity (GEMAS) study treated with ICS were analyzed. Control/case subjects were defined by the absence/presence of asthma exacerbations in the past 6 months despite being treated with ICS. The bacterial microbiota was profiled by sequencing the V3-V4 region of the 16S rRNA gene. Differences between groups were assessed by PERMANOVA and regression models adjusted for potential confounders. Afalse discovery rate (FDR) of 5% was used to correct for multiple comparisons. Classification models of asthma exacerbations despite ICS treatment were built with machine learning approaches based on clinical, genetic, and microbiome data. RESULTS: In nasal and saliva samples, case subjects had lower bacterial diversity (Richness, Shannon, and Faith indices) than control subjects (.007≤ P≤ .037). Asthma exacerbations accounted for 8% to 9% of the interindividual variation of the salivary and nasal microbiomes (.003≤ P≤ .046). Three, 4, and 11 bacterial genera from the salivary, pharyngeal, and nasal microbiomes were differentially abundant between groups (4.09*10-12≤ FDR≤ 0.047). Integrating clinical, genetic, and microbiome data showed good discrimination for the development of asthma exacerbations despite receipt of ICS (AUCtraining: 0.82 and AUCvalidation: 0.77). CONCLUSION: The diversity and composition of the upper-airway microbiome are associated with asthma exacerbations despite ICS treatment. The salivary microbiome has a potential application as a biomarker of asthma exacerbations despite receipt of ICS

Interacciones moleculares en la transferencia conjugativa del plásmido pMV158

La conjugación bacteriana es la transferencia de material genético desde uuna célula donadora a otra receptora mediante contacto físico. Por su alta capacidad de establecimiento en diversos patógenos Gram-positivos, pMV158 es un sistema interesante para el estudio del proceso conjugativo. Este plásmido contiene un origen de transferencia (oriT) yel fen mobM, que codifica la proteína iniciadora de la conjugación MobM. En el capítulo 1 se anaaliza la expresión del gen mobM. La RNA polimerasa de Escherichia coli reconoce como promotor una secuencia que solapa con el oriT, donde se une la proteína MobM. Así, la actividad relaxasa de MobM podría autorregular la expresión del gen mobM. El capítulo 2 estudia el dominio relaxasa de MobM. La versión MobMN199, que contiene los primeros 199 aminoácidos, mantiene la actividad relaxasa sobre DNA superenrollado. MobMN199 es un monómero, pesenta un alto contenido en hélice-α, alternando con cadena-β, y tiene alta afinidad por Mn 2+ , el cual aumenta la estabilidad térmica de la proteína. En el capítulo 3 se demuestra que la interacción de MobM con una estructura secundaria dentro del oriT genera un sustrato de DNA monocatenario, esencial para que se inicie la transferencia conjugativa. La definición de la secuencia mínima del oriT permitión la resolución de la estructura tridimensional de MobMN199, mostrando un plegamiento global y una estructura del centro activo similares al de otras relaxasas de estructura conocida. El capítulo 4 analiza el papel de los orígenes de cadena retrasada, ssoU y ssoA, en la transferencia de pMV158. Ambos ssos son funcionales en Streptococcus pneumoniae. Sin embargo, solo el origen ssoU es óptimo para la conjugación hacia Enterococcus faecalis. El modelo propuesto predice que la reconversión del DNA monocatenario a bicatenario de pMV158 se inicia desde uuuuuun sso de forma equivalente a la replicación vegetativa, dependiendo de la especie bacteriana receptora