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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 of 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 × 10³ transformants μg^-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 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 noncompetent 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 that 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.

SUBMITTER: Fonseca DR

PROVIDER: S-EPMC7549367 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Many surface structures in archaea including various types of pili and the archaellum (archaeal flagellum) are homologous to bacterial type IV pili systems (T4P). The T4P consist of multiple proteins, often with poorly conserved sequences, complicating their identification in sequenced genomes. Here we report a comprehensive census of T4P encoded in archaeal genomes using sensitive methods for protein sequence comparison. This analysis confidently identifies as T4P components about 5000 archaeal gene products, 56% of which are currently annotated as hypothetical in public databases. Combining results of this analysis with a comprehensive comparison of genomic neighborhoods of the T4P, we present models of organization of 10 most abundant variants of archaeal T4P. In addition to the differentiation between major and minor pilins, these models include extra components, such as S-layer proteins, adhesins and other membrane and intracellular proteins. For most of these systems, dedicated major pilin families are identified including numerous stand alone major pilin genes of the PilA family. Evidence is presented that secretion ATPases of the T4P and cognate TadC proteins can interact with different pilin sets. Modular evolution of T4P results in combinatorial variability of these systems. Potential regulatory or modulating proteins for the T4P are identified including KaiC family ATPases, vWA domain-containing proteins and the associated MoxR/GvpN ATPase, TFIIB homologs and multiple unrelated transcription regulators some of which are associated specific T4P. Phylogenomic analysis suggests that at least one T4P system was present in the last common ancestor of the extant archaea. Multiple cases of horizontal transfer and lineage-specific duplication of T4P loci were detected. Generally, the T4P of the archaeal TACK superphylum are more diverse and evolve notably faster than those of euryarchaea. The abundance and enormous diversity of T4P in hyperthermophilic archaea present a major enigma. Apparently, fundamental aspects of the biology of hyperthermophiles remain to be elucidated.

Project description:The rare actinomycete Actinoplanes missouriensis produces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili in A. missouriensis The pilus gene (pil) cluster, consisting of three apparently ?FliA-dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6?±?3 pili per zoospore (n?=?37 piliated zoospores) and a length of 0.62?±?0.35??m (n?=?206), via observation of fliC-deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encoding pilA deletion (?pilA) mutant. In addition, the deletion of pilT, which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and ?pilA zoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve the pil cluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth.IMPORTANCE Bacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomycete Actinoplanes missouriensis Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore.

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Type IV-Like Pili Facilitate Transformation in Naturally Competent Archaea.

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