Random transposon mutagenesis identifies genes essential for transformation in Methanococcus maripaludis

Abstract

Natural transformation, the process whereby a cell acquires DNA directly from the environment, is an important driver of evolution in microbial populations, yet the mechanism of DNA uptake is only characterized in bacteria. To expand our understanding of natural transformation in archaea, we undertook a genetic approach to identify a catalog of genes necessary for transformation in Methanococcus maripaludis. Using an optimized method to generate random transposon mutants, we screened 6144 mutant strains for defects in natural transformation and identified 25 transformation-associated candidate genes. Among these are genes encoding components of the type IV-like pilus, transcription/translation associated genes, genes encoding putative membrane bound transport proteins, and genes of unknown function. Interestingly, similar genes were identified regardless of whether replicating or integrating plasmids were provided as a substrate for transformation. Using allelic replacement mutagenesis, we confirmed that several genes identified in these screens are essential for transformation. Finally, we identified a homolog of a membrane bound substrate transporter in Methanoculleus thermophilus and verified its importance for transformation using allelic replacement mutagenesis, suggesting a conserved mechanism for DNA transfer in multiple archaea. These data represent an initial characterization of the genes important for transformation which will inform efforts to understand gene flow in natural populations. Additionally, knowledge of the genes necessary for natural transformation may assist in identifying signatures of transformation machinery in archaeal genomes and aid the establishment of new model genetic systems for studying archaea.

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen

“…al. (7, 39, 41) with slight differences in the starting culture preparation. Briefly, 16 overnight cultures in stationary phase (OD 600 ∼1.0) were supplemented with 1 mL fresh McCas and repressurized to 276 kPa H 2 :CO 2 and incubated shaking at 37°C for 2 hours.…”

“…Libraries were generated in two strains to account for a range of genotypes. The genomes of JJ and S2 are 95% identical (2-way average nucleotide identity) (6, 7), however they have major phenotypic differences. For example, strain JJ forms biofilms and can take up DNA via a natural transformation pathway in laboratory conditions while strain S2 exhibits more reliable growth in nitrogen fixing conditions.…”

Teaching old dogs new tricks: genetic engineering methanogens

“…In competent strains of M. maripaludis and M. thermophilus , type IV pilus is required for DNA uptake, and that competence may be conferred to a noncompetent M. maripaludis strain by expressing the pilin genes ( 70 ). A study using random mutagenesis identified genes necessary for natural transformation, which included genes encoding for type IV pilus, membrane-bound transporters, and other unknown genes ( 71 ).…”

“…Non-competent methanogens may require specific environmental conditions, such as quorum sensing cues, low substrate concentrations, or other stressors, to induce competence. For example, competence in M. maripaludis S2 must be induced by expressing pilin proteins, although M. maripaludis strain JJ is constitutively competent ( 71 ). The study of archaeal natural transformation is still a developing field; thus, new genetic tools may yield additional insights.…”

“…The mini-MAR367 element was then used to demonstrate the involvement of HrsM, a LysR-type transcriptional regulator, in the selenium-dependent gene expression of M. maripaludis ( 51 ). More recently, these transposon-mediated methods were used to identify the genes required for DNA uptake and transformation in M. maripaludis ( 71 ).…”