RESUMEN
Toxin-antitoxin (TA) systems are found on both chromosomes and plasmids. These systems are unique in that they can confer both fatal and protective effects on bacterial cells-a quality that could potentially be harnessed given further understanding of these TA mechanisms. The current work focuses on the ParE subfamily, which is found throughout proteobacteria and has a sequence identity on average of approximately 12% (similarity at 30%-80%). Our aim is to evaluate the equivalency of chromosomally derived ParE toxin activity depending on its bacterial species of origin. Nine ParE toxins were analyzed, originating from six different bacterial species. Based on the resulting toxicity, three categories can be established: ParE toxins that do not exert toxicity under the experimental conditions, toxins that exert toxicity within the first four hours, and those that exert toxicity only after 10-12 hr of exposure. All tested ParE toxins produce a cellular morphologic change from rods to filaments, consistent with disruption of DNA topology. Analysis of the distribution of filamented cells within a population reveals a correlation between the extent of filamentation and toxicity. No membrane septation is visible along the length of the cell filaments, whereas aberrant lipid blebs are evident. Potent ParE-mediated toxicity is also correlated with a hallmark signature of abortive DNA replication, consistent with the inhibition of DNA gyrase.
Asunto(s)
Topoisomerasa de ADN IV/biosíntesis , Topoisomerasa de ADN IV/toxicidad , Expresión Génica , Fenotipo , Proteobacteria/enzimología , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Topoisomerasa de ADN IV/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Mutágenos/metabolismo , Mutágenos/toxicidad , Conformación de Ácido Nucleico , Proteobacteria/citología , Proteobacteria/genética , Factores de TiempoRESUMEN
Bacterial cis-encoded antisense RNAs are transcribed from the opposite strand of protein coding genes, and their regulatory roles adapt cells to changing environmental conditions. By deep sequencing of the transcriptome of Salmonella enterica serovar Typhi, an antisense RNA that is encoded in cis to the parC gene was found. parC encodes the subunit A component of topoisomerase IV, a class of enzymes that relax both positively and negatively supercoiled DNA and are also required for segregation of daughter chromosomes in bacteria. Transcription of the 871 nucleotide antisense RNA was confirmed by northern blot and RACE analysis to be expressed mostly in the stationary phase of bacterial growth and also upregulated in iron limitation and osmotic stress conditions. Overexpression of the antisense RNA resulted in a significant increase in parC mRNA levels. Further analysis revealed that expression of the antisense RNA stabilizes the target mRNA, probably by protecting it from endoribonucleases. Our findings confirm and add to the ever increasing knowledge of the important role that regulatory antisense RNAs play in bacteria.
Asunto(s)
Topoisomerasa de ADN IV/biosíntesis , Topoisomerasa de ADN IV/genética , Regulación Bacteriana de la Expresión Génica , ARN sin Sentido/genética , Salmonella typhi/enzimología , Salmonella typhi/genética , Northern Blotting , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Estabilidad del ARNRESUMEN
The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥ 64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación/genética , Mutación/fisiología , Antibacterianos/farmacología , Área Bajo la Curva , Medios de Cultivo , Girasa de ADN/biosíntesis , Girasa de ADN/genética , Topoisomerasa de ADN IV/biosíntesis , Topoisomerasa de ADN IV/genética , Escherichia coli/efectos de los fármacos , Infecciones por Escherichia coli/microbiología , Eliminación de Gen , Levofloxacino , Pruebas de Sensibilidad Microbiana , Ofloxacino/farmacología , Reacción en Cadena de la Polimerasa , Quinolonas/farmacología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Fluoroquinolone-resistant isolates of Haemophilus influenzae, obtained from a long-term care facility, were examined for nucleotide sequence differences in the quinolone-resistance-determining regions of gyrA, gyrB, parC, and parE. Similarities among the resistant isolates, plus multiple differences with susceptible isolates, suggest clonal dissemination involving two resistant subclones.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Fluoroquinolonas/farmacología , Haemophilus influenzae/efectos de los fármacos , Instituciones de Cuidados Especializados de Enfermería , Girasa de ADN/biosíntesis , Topoisomerasa de ADN IV/biosíntesis , Farmacorresistencia Bacteriana , Genes Bacterianos , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences. The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system. The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively. Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains. In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B. subtilis.