Dataset Information

A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

ABSTRACT: New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.

SUBMITTER: Metcalf WW

PROVIDER: S-EPMC20139 | biostudies-literature | 1997 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

Metcalf W W WW Zhang J K JK Apolinario E E Sowers K R KR Wolfe R S RS

Proceedings of the National Academy of Sciences of the United States of America 19970301 6

New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allow ...[more]

PMID: 9122246

Similar Datasets

Project description:BackgroundThe plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.ResultsThe new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.ConclusionThe new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.

Project description:CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2 Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivoransIMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.

Dataset Information

A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

Publications

A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets