RESUMEN
Two A/C incompatibility group (IncA/C family) plasmids from the 1960s have been sequenced and classified into the A/C2 type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 genomic island not previously found in the family. In the 173.1-kb R16a, the 29.9-kb antibiotic resistance island (ARI) is located in a unique backbone position not utilized by ARIs. ARIR16a consists of Tn1, Tn6020, and Tn6333, harboring the resistance genes blaTEM-1D and aphA1b and a mer module, respectively; a truncated Tn5393 copy; and a gene cluster with unknown function. Plasmid IP40a is 170.4 kb in size and contains a 5.6-kb ARI inserted into the kfrA gene. ARIIP40a carrying blaTEM-1D and aphA1b genes is composed of Tn1 with a Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186, and Tn1000 insertions scattered in the backbone; an IS150 copy in GIsul2; and a complete Tn6333 carrying a mer module at the position of ARIR16a Loss of resistance markers in R16a, IP40a, and R55 was observed during stability tests. Every phenotypic change proved to be the result of recombination events involving mobile elements. Intramolecular transposition of IS copies that generated IP40a derivatives lacking large parts of the backbone could account for the formation of other family members, too. The MinION platform proved to be a valuable tool in bacterial genome sequencing since it generates long reads that span repetitive elements and facilitates full-length plasmid or chromosome assembly. Nanopore technology enables rapid characterization of large, low-copy-number plasmids and their rearrangement products.
Asunto(s)
ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Genoma Bacteriano , Plásmidos/química , Plásmidos/historia , beta-Lactamasas/genética , Antibacterianos/farmacología , Automatización de Laboratorios , Conjugación Genética , Elementos Transponibles de ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Islas Genómicas , Historia del Siglo XX , Plásmidos/metabolismo , Análisis de Secuencia de ADNRESUMEN
Plasmids are non-chromosomal hereditary determinants, mostly found in prokaryotes. Whereas Joshua Lederberg coined the term "plasmid" as early as 1952, today's concept was not established until the early 1970s. In this eclipse period, the plasmid's place was taken by the episome, following the 1958 publication of Elie Wollman and François Jacob. This paper analyzes the transition from the episome to a renewed plasmid concept both on the experimental and the conceptual level. It will become clear that intergeneric transfer experiments were central to this development. These studies rely on conjugational transfer of extrachromosomal hereditary determinants between different bacterial genera. First, experimental systems employing intergeneric transfer shaped the new plasmid by enabling its representation as a species of circular DNA. Moreover, they had a destabilizing effect on the episome, leading to a crisis in the concepts of microbial genetics towards the end of the 1960s. The new plasmid then became one of the cornerstones of recombinant DNA technologies. In an historic perspective, intergeneric transfer experiments indicate a gradual transition of molecular biology from its early "analytic" to the "synthetic" phase of genetic engineering. Hence, the construction of genetic hybrids in vivo as epitomized in the studies shown here marks an intermediate state that one could designate as "recombinant DNA avant la lettre".
Asunto(s)
Bacterias/genética , ADN Recombinante/historia , Hibridación Genética , Biología Molecular/historia , Plásmidos/historia , Conjugación Genética , Ingeniería Genética/historia , Investigación Genética/historia , Historia del Siglo XX , HumanosRESUMEN
pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most sensitive genetic toxicity assays and a useful short-term test for predicting carcinogenicity in animals. Plasmid pKM101, which is a deletion derivative of plasmid R46 (also referred to as R-Brighton after its origin of isolation in Brighton, England), has also been used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker's phenomenal insight into the genetics of Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis.
Asunto(s)
Pruebas de Carcinogenicidad/historia , Pruebas de Mutagenicidad/historia , Plásmidos/historia , Factores R/historia , Historia del Siglo XX , Mutagénesis , Plásmidos/genética , Plásmidos/aislamiento & purificación , Factores R/genética , Factores R/aislamiento & purificación , Salmonella/efectos de los fármacos , Salmonella/genéticaAsunto(s)
Agrobacterium tumefaciens/genética , ADN Bacteriano/historia , Plásmidos/historia , Agrobacterium tumefaciens/metabolismo , Enzimas de Restricción del ADN/historia , Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Ingeniería Genética/historia , Vectores Genéticos/historia , Historia del Siglo XX , Tumores de Planta/genética , Plantas/genética , Plantas/metabolismoRESUMEN
The term "plasmid" was introduced 45 years ago (J. Lederberg, 1952, Physiol. Rev. 32, 403-430) as a generic term for any extrachromosomal genetic particle. It was intended to clarify the classification of agents that had been thought of disjunctively as parasites, symbionts, organelles, or genes. For a decade or more it was confused with "episome," although that was carefully crafted (F. Jacob and E. L. Wollman, 1958, C. R. Acad. Sci. 247, 154-156) to mean agents with traffic in and out of chromosomes. Starting about 1970, plasmids became important reagents in molecular genetic research and biotechnology. They also play a cardinal role in the evolution of microbial resistance and of pathogenicity. The usage of the term has then escalated to its current peak of about 3000 published articles per year. The bedrock of genetic mechanism is no longer mitosis and meiosis of chromosomes; it is template-directed DNA assembly. This is often more readily studied and managed with the use of plasmids, which replicate autonomously outside the chromosomes. Some plasmids are also episomes, namely, they interact with the chromosomal genome, and other mobile elements may be transposed from one chromosomal locus to another without replicating autonomously.