A Novel Anchoring Mechanism for Prokaryotic Surface Proteins

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Archaea are prokaryotic organisms that are distinct from bacteria and although they are well known for the ability of many species to thrive in extreme environments, they are ubiquitous and can be found even in our gut and on our skin. Many cell surface proteins are essential to cellular functions (processes such as nutrient uptake, surface adhesion, and mating) and they must be effectively anchored to the cell surface in order to carry out their functions. In archaea, these attachment mechanisms are poorly understood and this project will characterize a novel surface protein anchoring mechanism that is also predicted to be used by many bacteria. The research project team will engage in science education to inspire an interest in science in young children, particularly those in underserved schools in West Philadelphia. The PI will develop microbiology experiments that can be utilized by schools with limited financial resources, and the researchers will participate in both in-class instruction and teacher-training workshops hosted by the University of Pennsylvania. In silico data suggest that many prokaryotes use a previously unknown mechanism to covalently link the C-terminus of a substrate to a lipid in the cell membrane. In archaea, this anchoring mechanism is likely catalyzed by an archaeosortase (ArtA) in a process that is reminiscent of the anchoring of surface proteins by the evolutionarily distinct bacterial sortase, an enzyme that processes and covalently links the C-termini of substrates to the cell wall. In vivo evidence supports the hypothesis that the Haloferax volcanii S-layer glycoprotein is processed and covalently linked to the lipid bilayer in an ArtA-dependent manner and has also revealed that other ArtA substrates play vital roles in important biological processes. The goal of the proposed research is to determine the specific roles that this ArtA-dependent anchoring mechanism plays in H. volcanii cellular processes, using biochemical, mass spectrometric, genetic and microscopic approaches. The proposed research will: 1) characterize post-translational modifications involving ArtA in greater detail; 2) determine structure-function relationships for ArtA, including the importance of a putative catalytic amino acid triad, as well as identifying other proteins that interact with ArtA; and 3) characterize the functional roles played by ArtA substrates in cellular processes such as mating, motility and adhesion.