Random transposon mutagenesis identifies genes essential for transformation in naturally competent archaea

Natural transformation, the process whereby a cell acquires DNA directly from the environment, is an important driver of evolution in microbial populations. While transformation is well characterized in bacteria, relatively little is known about this process in archaea. Here, we leverage an optimized method to generate transposon mutants in Methanococcus maripaludis to screen for genes essential to natural transformation. A screen of 5,376 mutant strains identified 25 candidate genes. Among these are genes encoding components of the type IV-like pilus, transcription/translation associated genes, 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 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 using allelic replacement mutagenesis, suggesting a conserved mechanism for DNA transfer in multiple archaea. These data provide an initial catalog of genes important for transformation in the archaea and can inform efforts to understand gene flow in this domain. Importance Horizontal gene transfer (HGT) is an important driver of evolution in microbial populations. One of the primary ways microorganisms acquire genetic material through HGT is transformation, the direct uptake of DNA from the environment. While transformation is well-studied in bacteria, little is known about this process in archaea. Using a random mutagenesis screen to identify components of the archaeal transformation pathway, we identify a catalog of genes important to transformation in Methanococcus maripaludis and show that a subset of these genes is functionally conserved across diverse archaea. This is a key step in understanding mechanisms of gene flow in natural populations, and identification of the DNA uptake system will assist in establishing new model genetic systems for studying the archaea.