RESUMEN
Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.
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Human exposure to organic mercury (Hg) as methylmercury (MeHg) from seafood consumption is widely considered a health risk because pure methylmercury is extremely neurotoxic. In contrast, the clinical significance of Hg exposure from amalgam (AMG) dental restorations, the only other major nonoccupational source of Hg exposure, has long been debated. Here, we examined data from the two most recent National Health and Nutrition Examination Surveys (NHANES) on 14 181 subjects to assess the contributions of seafood consumption versus AMG to blood total mercury (THg), inorganic mercury (IHg), and methyl mercury (MeHg) and to urine creatinine corrected mercury (UTHg). All subjects were also classified as to their self-reported qualitative consumption of seafood (59% fish and 44% shellfish). Subjects with restorations were grouped into three groups (0) those without AMG (64.4%), (1) those with 1-5 dental AMG restorations (19.7%), (2) those with more than five AMG (16%). Seafood consumption increased total mercury in urine (UTHg) and total mercury (THg) and methyl mercury (MeHg) in blood, but unlike AMG, seafood did not increase blood inorganic mercury (IHg). Using stratified covariate (ANOVA) and multivariate (GLM) analyses revealed a strong correlation of blood (THg and IHg) and urine (UTHg) levels with the number of AMGs. In a subpopulation without fish consumption, having more than five AMG restorations raised blood THg (103%), IHg (221%), and urine UTHg (221%) over the group without AMG. The most striking difference was noted in classification by age: subjects under 6 years old with more than five AMG restorations had the highest blood IHg and urine UTHg among all age groups. Elevation of bivalent IHg on a large scale in children warrants urgent in-depth risk assessment with specific attention to genetic- and gender-associated vulnerabilities.
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Mercurio , Compuestos de Metilmercurio , Animales , Peces , Humanos , Encuestas Nutricionales , Alimentos Marinos/análisisRESUMEN
The compounds of mercury can be highly toxic and can interfere with a range of biological processes, although many aspects of the mechanism of toxicity are still obscure or unknown. One especially intriguing property of Hg(II) is its ability to bind DNA directly, making interstrand cross-links between thymine nucleobases in AT-rich sequences. We have used a combination of small molecule X-ray diffraction, X-ray spectroscopies, and computational chemistry to study the interactions of Hg(II) with thymine. We find that the energetically preferred mode of thymine binding in DNA is to the N3 and predict only minor distortions of the DNA structure on binding one Hg(II) to two cross-adjacent thymine nucleotides. The preferred geometry is predicted to be twisted away from coplanar through a torsion angle of between 32 and 43°. Using 1-methylthymine as a model, the bis-thymine coordination of Hg(II) is found to give a highly characteristic X-ray spectroscopic signature that is quite distinct from other previously described biological modes of binding of Hg(II). This work enlarges and deepens our view of significant biological targets of Hg(II) and demonstrates tools that can provide a characteristic signature for the binding of Hg(II) to DNA in more complex matrices including intact cells and tissues, laying the foundation for future studies of mechanisms of mercury toxicity.
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ADN/química , Mercurio/química , Timina/química , Sitios de Unión , Teoría Funcional de la DensidadRESUMEN
Multi-antibiotic resistant (MAR) bacteria cost billions in medical care and tens of thousands of lives annually but perennial calls to limit agricultural and other misuse of antibiotics and to fund antibiotic discovery have not slowed this MAR deluge. Since mobile genetic elements (MGEs) stitch single antibiotic resistance genes into clinically significant MAR arrays, it is high time to focus on how MGEs generate MAR and how disabling them could ameliorate the MAR problem. However, to consider only antibiotics as the drivers of MAR is to miss the significant impact of exposure to non-antibiotic toxic chemicals, specifically metals, on the persistence and spread of MAR. Toxic metals were among the earliest discovered targets of plasmid-encoded resistance genes. Recent genomic epidemiology clearly demonstrated the co-prevalence of metal resistances and antibiotic multi-resistance, uniquely in humans and domestic animals. Metal resistances exploit the same, ancient "transportation infrastructure" of plasmids, transposons, and integrons that spread the antibiotic resistance genes and will continue to do so even if all antibiotic misuse were stopped today and new antibiotics were flowing from the pipeline monthly. In a key experiment with primates, continuous oral exposure to mercury (Hg) released from widely used dental amalgam fillings co-selected for MAR bacteria in the oral and fecal commensal microbiomes and, most importantly, when amalgams were replaced with non-metal fillings, MAR bacteria declined dramatically. Could that also be happening on the larger public health scale as use of amalgam restorations is curtailed or banned in many countries? This commentary covers salient past and recent findings of key metal-antibiotic resistance associations and proposes that the shift from phenotyping to genotyping in surveillance of resistance loci will allow a test of whether declining exposure to this leading source of Hg is accompanied by a decline in MAR compared to countries where amalgam is still used. If this hypothesis is correct, the limited success of antibiotic stewardship practices may be because MAR is also being driven by continuous, daily exposure to Hg, a non-antibiotic toxicant widely used in humans.
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Bacterias/genética , Farmacorresistencia Bacteriana Múltiple/genética , Secuencias Repetitivas Esparcidas/genética , Plásmidos/genética , Programas de Optimización del Uso de los Antimicrobianos , Bacterias/efectos de los fármacos , Bacterias/patogenicidad , Amalgama Dental/toxicidad , Humanos , Secuencias Repetitivas Esparcidas/efectos de los fármacos , Mercurio/toxicidad , Metales/toxicidadRESUMEN
After publication of the original article [1] the authors noted that the key displayed in Figure 1a was incorrect, as the PMA and HgCl2 conditions had been switched.
RESUMEN
BACKGROUND: The protean chemical properties of mercury have long made it attractive for diverse applications, but its toxicity requires great care in its use, disposal, and recycling. Mercury occurs in multiple chemical forms, and the molecular basis for the distinct toxicity of its various forms is only partly understood. Global transcriptomics applied over time can reveal how a cell recognizes a toxicant and what cellular subsystems it marshals to repair and recover from the damage. The longitudinal effects on the transcriptome of exponential phase E. coli were compared during sub-acute exposure to mercuric chloride (HgCl2) or to phenylmercuric acetate (PMA) using RNA-Seq. RESULTS: Differential gene expression revealed common and distinct responses to the mercurials throughout recovery. Cultures exhibited growth stasis immediately after each mercurial exposure but returned to normal growth more quickly after PMA exposure than after HgCl2 exposure. Correspondingly, PMA rapidly elicited up-regulation of a large number of genes which continued for 30 min, whereas fewer genes were up-regulated early after HgCl2 exposure only some of which overlapped with PMA up-regulated genes. By 60 min gene expression in PMA-exposed cells was almost indistinguishable from unexposed cells, but HgCl2 exposed cells still had many differentially expressed genes. Relative expression of energy production and most metabolite uptake pathways declined with both compounds, but nearly all stress response systems were up-regulated by one or the other mercurial during recovery. CONCLUSIONS: Sub-acute exposure influenced expression of ~45% of all genes with many distinct responses for each compound, reflecting differential biochemical damage by each mercurial and the corresponding resources available for repair. This study is the first global, high-resolution view of the transcriptional responses to any common toxicant in a prokaryotic model system from exposure to recovery of active growth. The responses provoked by these two mercurials in this model bacterium also provide insights about how higher organisms may respond to these ubiquitous metal toxicants.
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Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/genética , Cloruro de Mercurio/toxicidad , Acetato Fenilmercúrico/toxicidad , Transcriptoma/efectos de los fármacos , Transporte de Electrón/genética , Escherichia coli K12/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
The protean chemical properties of the toxic metal mercury (Hg) have made it attractive in diverse applications since antiquity. However, growing public concern has led to an international agreement to decrease its impact on health and the environment. During a recent proteomics study of acute Hg exposure in E. coli, we also examined the effects of inorganic and organic Hg compounds on thiol and metal homeostases. On brief exposure, lower concentrations of divalent inorganic mercury Hg(II) blocked bulk cellular thiols and protein-associated thiols more completely than higher concentrations of monovalent organomercurials, phenylmercuric acetate (PMA) and merthiolate (MT). Cells bound Hg(II) and PMA in excess of their available thiol ligands; X-ray absorption spectroscopy indicated nitrogens as likely additional ligands. The mercurials released protein-bound iron (Fe) more effectively than common organic oxidants and all disturbed the Na(+)/K(+) electrolyte balance, but none provoked efflux of six essential transition metals including Fe. PMA and MT made stable cysteine monothiol adducts in many Fe-binding proteins, but stable Hg(II) adducts were only seen in CysXxx(n)Cys peptides. We conclude that on acute exposure: (a) the distinct effects of mercurials on thiol and Fe homeostases reflected their different uptake and valences; (b) their similar effects on essential metal and electrolyte homeostases reflected the energy dependence of these processes; and (c) peptide phenylmercury-adducts were more stable or detectable in mass spectrometry than Hg(II)-adducts. These first in vivo observations in a well-defined model organism reveal differences upon acute exposure to inorganic and organic mercurials that may underlie their distinct toxicology.
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Escherichia coli/efectos de los fármacos , Homeostasis/efectos de los fármacos , Proteínas de Unión a Hierro/metabolismo , Mercurio/farmacología , Mercurio/toxicidad , Espectroscopía de Resonancia por Spin del Electrón , Contaminantes Ambientales/toxicidad , Compuestos Organomercuriales/toxicidad , Compuestos de SulfhidriloRESUMEN
We have developed a thiol-modified nanoporous silica material (SH-SAMMS) as an oral therapy for the prevention and treatment of heavy metal poisoning. SH-SAMMS has been reported to be highly efficient at capturing heavy metals in biological fluids and water. Herein, SH-SAMMS was examined for efficacy and safety in both in vitro and in vivo animal models for the oral detoxification of heavy metals. In simulated gastrointestinal fluids, SH-SAMMS had a very high affinity (Kd) for methyl mercury (MeHg(I)), inorganic mercury (Hg(II)), lead (Pb(II)), and cadmium (Cd(II)) and was superior to other SAMMS with carboxylic acid or phosphonic acid ligands or commercially available metal chelating sorbents. SH-SAMMS also effectively removed Hg from biologically digested fish tissue with no effect on most nutritional minerals found in fish. SH-SAMMS could hold Hg(II) and MeHg(I) tightly inside the nanosize pores, thus preventing bacteria from converting them to more absorbable forms. Rats fed a diet containing MeHg(I), Cd(II), and Pb(II) and SH-SAMMS for 2 weeks had blood Hg levels significantly lower than rats fed the metal-rich diet only. Upon cessation of the metal-rich diet, continued administration of SH-SAMMS for 2 weeks facilitated faster and more extensive clearance of Hg than in animals not continued on oral SH-SAMMS. Rats receiving SH-SAMMS also suffered less weight loss as a result of the metal exposure. Retention of Hg and Cd in major organs was lowest in rats fed with SH-SAMMS throughout the entire four weeks. The reduction of blood Pb by SH-SAMMS was significant. SH-SAMMS was safe to intestinal epithelium model (Caco-2) and common intestinal bacteria (Escherichia coli). Altogether, it has great potential as a new oral drug for the treatment of heavy metal poisoning. This new application is enabled by the installation of tailored interfacial chemistry upon nontoxic nanoporous materials.
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Cadmio/química , Quelantes/administración & dosificación , Terapia por Quelación/métodos , Intoxicación por Metales Pesados , Plomo/química , Mercurio/química , Intoxicación/tratamiento farmacológico , Dióxido de Silicio/administración & dosificación , Adsorción , Animales , Células CACO-2 , Cadmio/toxicidad , Quelantes/química , Terapia por Quelación/instrumentación , Humanos , Riñón/química , Riñón/efectos de los fármacos , Cinética , Plomo/toxicidad , Hígado/química , Hígado/efectos de los fármacos , Masculino , Mercurio/toxicidad , Ratas Wistar , Dióxido de Silicio/química , Compuestos de Sulfhidrilo/químicaRESUMEN
Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (≥30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes.
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Escherichia coli/genética , Variación Genética , Plásmidos/genética , Replicón , Salmonella/genética , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Secuencia Conservada , ADN Bacteriano/genética , Evolución Molecular , Genes Bacterianos , Genotipo , Secuencias Repetitivas Esparcidas , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The integrase IntI1 catalyses recombination of antibiotic-resistance gene cassettes in the integron, a widely found bacterial mobile element active in spreading antibiotic multi-resistance. We have previously shown that resistance cassette recombination rate and specificity depend on the amount of intracellular integrase. Here, we used in vivo and in vitro methods to examine convergent expression of the integrase promoter (P(int)) and of the cassette promoters (P(c) and P(2)) in the prototypical plasmid-borne class 1 integron, In2. Highly conserved P(int) has near consensus -10 and -35 hexamers for σ(70) RNA polymerase, but there are 11 naturally occurring arrangements of P(c) alone or combinations of the P(c)+P(2) cassette promoters (note that P(2) occurs with a 14 or 17 bp spacer). Using a bi-directional reporter vector, we found that P(int) is a strong promoter in vivo, but its expression is reduced by converging transcription from P(c) and P(2). In addition to cis-acting convergence control of integrase expression, the regulator site prediction program, prodoric 8.9, identified sites for global regulators FIS, LexA, IHF and H-NS in and near the integron promoters. In strains mutated in each global regulator, we found that: (1) FIS repressed integrase and cassette expression; (2) LexA repressed P(int) and P(2) with the 14 bp spacer version of P(2) and FIS was necessary for maximum LexA repression; (3) IHF activated P(int) when it faced the strong 17 bp spacer P(2) but did not elevate its expression versus LexA-repressed P(2) with the 14 bp spacer; and (4) H-NS repressed both P(int) and the 14 bp P(2) but activated the 17 bp P(2) cassette promoters. Mobility shift assays showed that FIS and IHF interact directly with the promoter regions and DNase I footprinting confirmed extensive protection by FIS of wild-type In2 integron promoter sequence. Thus, nucleoid-associated proteins, known to act directly in site-specific recombination, also control integron gene expression directly and possibly indirectly.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica , Integrasas/genética , Integrones , Regiones Promotoras Genéticas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Integrasas/metabolismo , Unión Proteica , Transcripción GenéticaRESUMEN
The identification of peptides that result from post-translational modifications is critical for understanding normal pathways of cellular regulation as well as identifying damage from, or exposures to xenobiotics, i.e. the exposome. However, because of their low abundance in proteomes, effective detection of modified peptides by mass spectrometry (MS) typically requires enrichment to eliminate false identifications. We present a new method for confidently identifying peptides with mercury (Hg)-containing adducts that is based on the influence of mercury's seven stable isotopes on peptide isotope distributions detected by high-resolution MS. Using a pure protein and E. coli cultures exposed to phenyl mercuric acetate, we show the pattern of peak heights in isotope distributions from primary MS single scans efficiently identified Hg adducts in data from chromatographic separation coupled with tandem mass spectrometry with sensitivity and specificity greater than 90%. Isotope distributions are independent of peptide identifications based on peptide fragmentation (e.g. by SEQUEST), so both methods can be combined to eliminate false positives. Summing peptide isotope distributions across multiple scans improved specificity to 99.4% and sensitivity above 95%, affording identification of an unexpected Hg modification. We also illustrate the theoretical applicability of the method for detection of several less common elements including the essential element, selenium, as selenocysteine in peptides.
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Isótopos de Mercurio/química , Procesamiento Proteico-Postraduccional , Proteoma/química , Algoritmos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Escherichia coli , Proteínas de Escherichia coli/química , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Marcaje Isotópico , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteómica , Conejos , Relación Señal-Ruido , Espectrometría de Masas en TándemRESUMEN
Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20-30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.
RESUMEN
USA300 methicillin-resistant Staphylococcus aureus (MRSA) isolates are usually resistant only to oxacillin, erythromycin, and, increasingly, levofloxacin. Of these, oxacillin and levofloxacin resistances are chromosomally encoded. Plasmid-mediated clindamycin, mupirocin, and/or tetracycline resistance has been observed among USA300 isolates, but these descriptions were limited to specific patient populations or isolated occurrences. We examined the antimicrobial susceptibilities of invasive MRSA isolates from a national surveillance population in order to identify USA300 isolates with unusual, possibly emerging, plasmid-mediated antimicrobial resistance. DNA from these isolates was assayed for the presence of resistance determinants and the presence of a pSK41-like conjugative plasmid. Of 823 USA300 isolates, 72 (9%) were tetracycline resistant; 69 of these were doxycycline susceptible and tetK positive, and 3 were doxycycline resistant and tetM positive. Fifty-one (6.2%) isolates were clindamycin resistant and ermC positive; 22 (2.7%) isolates were high-level mupirocin resistant (mupA positive); 5 (0.6%) isolates were trimethoprim-sulfamethoxazole (TMP-SMZ) resistant, of which 4 were dfrA positive; and 7 (0.9%) isolates were gentamicin resistant and aac6'-aph2'' positive. Isolates with pSK41-like plasmids (n = 24) were positive for mupA (n = 19), dfrA (n = 6), aac6'-aph2'' (n = 6), tetM (n = 2), and ermC (n = 8); 20 pSK41-positive isolates were positive for two or more resistance genes. Conjugative transfer of resistance was demonstrated between four gentamicin- and mupirocin-resistant and three gentamicin- and TMP-SMZ-resistant USA300 isolates; transconjugants harbored a single pSK41-like plasmid, which was PCR positive for aac6'-aph2'' and either mupA and/or dfrA. USA300 and USA100 isolates from the same state with identical resistance profiles contained pSK41-like plasmids with indistinguishable restriction and Southern blot profiles, suggesting horizontal plasmid transfer between USA100 and USA300 isolates.
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Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Clindamicina/farmacología , Doxiciclina/farmacología , Resistencia a Múltiples Medicamentos , Gentamicinas/farmacología , Staphylococcus aureus Resistente a Meticilina/genética , Pruebas de Sensibilidad Microbiana , Mupirocina/farmacología , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Tetraciclina/farmacología , Combinación Trimetoprim y Sulfametoxazol/farmacología , Estados UnidosRESUMEN
The bacterial metalloregulator MerR is the index case of an eponymous family of regulatory proteins, which controls the transcription of a set of genes (the mer operon) conferring mercury resistance in many bacteria. Homodimeric MerR represses transcription in the absence of mercury and activates transcription upon Hg(II) binding. Here, the average structures of the apo and Hg(II)-bound forms of MerR in aqueous solution are examined using small-angle X-ray scattering, indicating an extended conformation of the metal-bound protein and revealing the existence of a novel compact conformation in the absence of Hg(II). Molecular dynamics (MD) simulations are performed to characterize the conformational dynamics of the Hg(II)-bound form. In both small-angle X-ray scattering and MD, the average torsional angle between DNA-binding domains is approximately 65 degrees. Furthermore, in MD, interdomain motions on a timescale of approximately 10 ns involving large-amplitude (approximately 20 A) domain opening-and-closing, coupled to approximately 40 degrees variations of interdomain torsional angle, are revealed. This correlated domain motion may propagate allosteric changes from the metal-binding site to the DNA-binding site while maintaining DNA contacts required to initiate DNA underwinding.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Mercurio/metabolismo , Regulación Alostérica , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Dispersión del Ángulo PequeñoRESUMEN
Demethylation is a key reaction in global mercury cycling. The bacterial organomercurial lyase, MerB, catalyzes the demethylation of a wide range of organomercurials via Hg-C protonolysis. Two strictly conserved cysteine residues in the active site are required for catalysis, but the source of the catalytic proton and the detailed reaction mechanism have not been determined. Here, the two major proposed reaction mechanisms of MerB are investigated and compared using hybrid density functional theory calculations. A model of the active site was constructed from an X-ray crystal structure of the Hg(II)-bound MerB product complex. Stationary point structures and energies characterized for the Hg-C protonolysis of methylmercury rule out the direct protonation mechanism in which a cysteine residue delivers the catalytic proton directly to the organic leaving group. Instead, the calculations support a two-step mechanism in which Cys96 or Cys159 first donates a proton to Asp99, enabling coordination of two thiolates with R-Hg(II). At the rate-limiting transition state, Asp99 protonates the nascent carbanion in a trigonal planar, bis thiol-ligated R-Hg(II) species to cleave the Hg-C bond and release the hydrocarbon product. Reactions with two other substrates, vinylmercury and cis-2-butenyl-2-mercury, were also modeled, and the computed activation barriers for all three organomercurial substrates reproduce the trend in the experimentally observed enzymatic reaction rates. Analysis of atomic charges in the rate-limiting transition state structure using Natural Population Analysis shows that MerB lowers the activation free energy in the Hg-C protonolysis reaction by redistributing electron density into the leaving group and away from the catalytic proton.
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Carbono/metabolismo , Liasas/metabolismo , Mercurio/metabolismo , Modelos Moleculares , Biocatálisis , Dominio Catalítico , Liasas/química , Teoría Cuántica , Agua/metabolismoRESUMEN
Some elements are essential for life and others closely related to them are very toxic. In exploiting unique ecological niches many prokaryotes have evolved the means to defend themselves against and even to derive energy from deleterious elements. Toxic metal defense systems are related to those providing homeostasis of essential metals and metalloid elements. Expression of these multiprotein systems is costly but they must respond rapidly and, so, all are well controlled. Seven diverse families of metalloregulators are presently recognized for essential metal homeostasis in prokaryotes. Two of these, the ArsR and MerR families, figure more often than the others in controlling responses to toxic transition metals and metalloids. This review emphasizes recent advances in these two metalloregulator families and highlights emerging regulatory motifs of other types.
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Bacterias/metabolismo , Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Metales/metabolismo , Metales/toxicidad , Transactivadores/fisiología , Bacterias/efectos de los fármacos , HomeostasisRESUMEN
IntI1 mediates the recombination of antibiotic-resistant gene cassettes between different integrons in the same cell, facilitating the persistence and dissemination of these genes. Historically, integrase activity has been measured by conjugating recombinant products from donor cells overexpressing integrase and quantifying them in recipient cells. Here we report the first measurements of the steady-state intracellular abundance of integrase-mediated recombination products in strains expressing natural or high IntI1 levels. Recombination products in both high and natural integrase strains increased markedly through late log phase and continued to rise in stationary phase in the high integrase strain, but declined in the natural expression strain. Simple acquisition of gene cassettes was seen only in strains expressing high integrase; in strains with natural integrase levels, only cointegrates between the two integron-bearing plasmids were detectable at all growth stages. Unexpectedly, more attIxattI than attCxattI recombination products were seen in log phase for both strains; however, in stationary phase, the high integrase strain increased attC recombination, consistent with earlier observations of integrase crossover site preferences. Thus, direct quantification of the steady-state concentration of recombination products reveals that the integrase's intracellular concentration affects the amount and type of recombination events in a growth-phase-dependent manner.
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Fenómenos Fisiológicos Bacterianos , Ciclo Celular , ADN Bacteriano/metabolismo , Integrasas/metabolismo , Integrones , Recombinación Genética , ADN Bacteriano/genéticaRESUMEN
Transposable DNA elements occur naturally in the genomes of nearly all species of prokaryotes. A proposal for a uniform transposable element nomenclature was published prominently in the 1970s but is not, at present, available online even in abstract form, and many of the newly discovered elements have been named without reference to it. We propose here an updated version of the original nomenclature system for all of the various types of prokaryotic, autonomous, transposable elements excluding insertion sequences, for which a nomenclature system already exists. The use of this inclusive and sequential Tn numbering system for transposable elements, as described here, recognizes the ease of interspecies spread of individual elements, and allows for the naming of mosaic elements containing segments from two or more previously described types of transposons or plasmids. It will guard against any future need to rename elements following changes in bacterial nomenclature which occurs constantly with our increased understanding of bacterial phylogenies and taxonomic groupings. It also takes into account the increasing importance of metagenomic sequencing projects and the continued identification of new mobile elements from unknown hosts.
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Elementos Transponibles de ADN/genética , Terminología como AsuntoRESUMEN
Metalloregulators of the MerR family activate transcription upon metal binding by underwinding the operator-promoter DNA to permit open complex formation by pre-bound RNA polymerase. Historically, MerR's allostery has been monitored only indirectly via nuclease sensitivity or by fluorescent nucleotide probes and was very specific for Hg(II), although purified MerR binds several thiophilic metals. To observe directly MerR's ligand-induced behavior we made 2-fluorotyrosine-substituted MerR and found similar, minor changes in (19)F chemical shifts of tyrosine residues in the free protein exposed to Hg(II), Cd(II) or Zn(II). However, DNA binding elicits large chemical shift changes in MerR's tyrosine residues and in DNA-bound MerR Hg(II) provokes changes very distinct from those of Cd(II) or Zn(II). These chemical shift changes and other biophysical and phenotypic properties of wild-type MerR and relevant mutants reveal elements of an allosteric network that enables the coordination state of the metal binding site to direct metal-specific movements in the distant DNA binding site and the DNA-bound state also to affect the metal binding domain.