RESUMEN
The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing.
Asunto(s)
Antiinfecciosos/administración & dosificación , Proteína 9 Asociada a CRISPR/administración & dosificación , Conjugación Genética , Sistemas de Liberación de Medicamentos/métodos , Técnicas de Transferencia de Gen , Biopelículas/efectos de los fármacos , Proteína 9 Asociada a CRISPR/genética , Técnicas de Cocultivo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , ARN Guía de Kinetoplastida/genética , Saccharomyces cerevisiae , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genéticaRESUMEN
Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowiaborealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metschnikowia species, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 21 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications.
Asunto(s)
Técnicas de Transferencia de Gen , Transformación Genética , Levaduras/genética , Codón/genética , Electroporación/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P. tricornutum promoters and terminators allowing selective expression of antibiotic resistance markers from stably replicating plasmids in P. tricornutum. Significantly, we developed a protocol for sequential conjugation of plasmids from Escherichia coli to P. tricornutum and demonstrated simultaneous replication of two plasmids in P. tricornutum. We developed a simple and robust conjugative system for Cas9 editing that yielded up to 60% editing efficiency of the urease gene. Finally, we constructed a plasmid encoding eight genes involved in vanillin biosynthesis that was propagated in P. tricornutum over four months with no evidence of rearrangements, with whole-plasmid sequencing indicating that the majority of mutations occurred after plasmid assembly and initial conjugation rather than during long-term propagation. The plasmid-based tools described here will facilitate investigation of the basic biology of P. tricornutum and enable synthetic biology applications.
