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.

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

In the environment, methane is typically generated by fermentative bacteria and methanogenic archaea working together in a process called syntrophy. Efficient exchange of small molecules like H 2 or formate is essential for growth of both organisms.

Most microbial organisms grow as surface-attached communities known as biofilms. However, the mechanisms whereby methanogenic archaea grow attached to surfaces have remained understudied. Here, we show that the oligosaccharyltransferase AglB is essential for growth of Methanococcus maripaludis strain JJ on glass or metal surfaces. AglB glycosylates several cellular structures such as pili, archaella, and the cell surface layer (S-layer). We show that the S-layer of strain JJ, but not strain S2, is a glycoprotein, that only strain JJ was capable of growth on surfaces, and that deletion of aglB blocked S-layer glycosylation and abolished surface-associated growth. A strain JJ mutant lacking structural components of the type IV-like pilus did not have a growth defect under any conditions tested, while a mutant lacking the pre-flagellin peptidase (Δ flaK ) was only defective for surface growth when formate was provided as the sole electron donor. Finally, for strains that are capable of Fe 0 oxidation, we show that deletion of aglB decreases the rate of anaerobic Fe 0 oxidation, presumably due to decreased association of biomass with the Fe 0 surface. Together, these data provide an initial characterization of surface-associated growth in a member of the methanogenic archaea. Importance Methanogenic archaea are responsible for producing the majority of methane on Earth and catalyze the terminal reactions in the degradation of organic matter in anoxic environments. Methanogens often grow as biofilms associated with surfaces or partner organisms; however, the molecular details of surface-associated growth remain uncharacterized. We have found evidence that glycosylation of the cell surface layer is essential for growth of M. maripaludis on surfaces and can enhance rates of anaerobic iron corrosion. These results provide insight into the physiology of surface-associated methanogenic organisms and highlight the importance of surface association to anaerobic iron corrosion.

Enzyme‐catalyzed β‐lactone formation from β‐hydroxy acids is a crucial step in bacterial biosynthesis of β‐lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β‐lactone synthetase activity using synthetic β‐hydroxy acid substrates with alkene or alkyne moieties. β‐Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real‐time evaluation by UV/Vis spectroscopy. The assay was used to determine steady‐state kinetics of a long‐chain β‐lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site‐directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the β‐lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC‐MS. Stereoisomers of 2‐hexyl‐3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo‐preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the β‐lactone synthetases to map their functional diversity within the acyl‐CoA synthetase, NRPS adenylation domain, and luciferase superfamily.