Type IV-Like Pili Facilitate Transformation in Naturally Competent Archaea

Abstract

Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms: Methanococcus maripaludis and Methanoculleus thermophilus. In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 x 103 transformants μg-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 x 103 transformants μg-1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales. For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a non-competent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms. Importance Microbial organisms adapt and evolve by acquiring new genetic material through horizontal gene transfer. One way this occurs is natural transformation, the direct uptake and genomic incorporation of environmental DNA by competent organisms. Archaea represent up to a third of the biodiversity on Earth, yet little is known about transformation in these organisms. Here we provide the first characterization of a component of the archaeal DNA uptake machinery. We show that the type IV-like pilus is essential for natural transformation in two archaeal species. This suggests that pili are important for transformation across the tree of life and further expands our understanding of gene flow in archaea.

A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH

“…For both antibiotics, thermostable selectable markers are available, which have been successfully used in thermophilic nonmethanogenic microbes, such as Pyrococcus furiosus and Thermococcus kodakarensis (simvastatin) ( 21 , 22 ), as well as Thermoanaerobacter spp. (neomycin) ( 23 ), but recently also in the thermophilic methanogens Methanocaldococcus jannaschii (simvastatin) and Methanoculleus thermophilus (neomycin) ( 24 , 25 ). Both simvastatin and neomycin efficiently inhibited growth of M. thermautotrophicus ΔH cells in liquid culture at concentrations of 21.5 μg/ml and 250 μg/ml, respectively ( Text S1B and Fig.…”

A shuttle-vector system allows heterologous gene expression in the thermophilic methanogenMethanothermobacter thermautotrophicusΔH

“…(neomycin) (36), but recently also in the thermophilic methanogens M. jannaschii (simvastatin) and M. thermophilus (neomycin) (18,26) To ease exchangeability of genetic elements in shuttle vectors, and to allow fast adaptation to new findings, we decided for a modular plasmid design from the beginning. Inspired by the pSEVA system for Gram-negative bacteria (37), and the pMTL80000 system for Clostridia (38), we established the Based on the first shuttle vector (pMVS-V1) that led to successful DNA transfer and selection protocols, as described in the next section, we defined pMVS-V1 as our archetype shuttle vector (Figure 1A), with a combination of: 1) the ColE1derived replicon for E. coli in combination with the conjugational transfer function Following our broad approach, we investigated several published protocols (and modified versions) to transfer DNA into M. thermautotrophicus Δ H by using our various plasmids and shuttle vectors (Material and Methods).…”

“…and Methanosarcina spp.. Sophisticated genetic tools for these microbes have been implemented, such as allelic exchange methods and counter-selection markers to generate gene deletion strains, and recently CRISPR/Cas9-based genome engineering (15)(16)(17). The transfer of DNA into the cells for these systems has been achieved with varying efficiencies via: 1) natural competence (18); 2) polyethylene glycol-mediated transformation of protoplasts (16); 3) liposome-mediated transformation of protoplasts (19); 4) electroporation (20); and 5) conjugation (21). As antibioticselectable markers, a puromycin-selectable marker (19), different kanamycin/neomycin-selectable markers (22), a mupirocin-selectable marker (12), and a nourseothricin-selectable marker (23) have been implemented.…”

“…In addition to the mesophilic systems, genetic tools for the thermophilic methanogens Methanocaldococcus jannaschii and Methanoculleus thermophilus have recently been described (18,26). DNA transfer was achieved via heat shock-mediated transformation (M. jannaschii) or natural competence (M. thermophilus).…”

“…DNA transfer was achieved via heat shock-mediated transformation (M. jannaschii) or natural competence (M. thermophilus). Positive selection with antibiotics was accomplished for M. jannaschii with a simvastatin/mevilonin-selectable marker under the control of the native constitutive promoter of an S-Layer protein-encoding gene (P Sla(M.j.) ) (26), and for M. thermophilus with a thermostability-adapted neomycin-selectable marker (27) under the control of the promoter of the methyltetrahydromethanopterin:coenzyme M methyl-transferase (mtr) operon from M. jannaschii (P mtr(M.j.) ) (18). Both systems rely on the integration of DNA into the genome by homologous recombination, and replicating plasmid vectors for the thermophilic methanogens have not been described.…”