Lipid Anchoring of Archaeosortase Substrates and Mid-Cell Growth in Haloarchaea

Abstract

The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA) and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and mor-phogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. H. volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth and morphological phenotypes similar to those of ∆artA. Moreover, we showed the loss of covalent lipid attachment to SLG in the ∆hvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ∆hvpssA and ∆hvpssD strains. Strikingly, ArtA, HvPssA, and HvPssD GFP-fusions co-localized to the mid position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the mid cell prior to being secreted and lipid-anchored at the cell outer surface. Collectively, our data suggest haloarchaea use the mid cell as the main surface processing hotspot for cell elongation, division and shape determination.

Molecular Logic of Prokaryotic Surface Layer Structures

“…In species of the order Methanobacteriales (e.g., M. fervidus), however, the cytoplasmic membrane is surrounded by a cell wall composed of pseudopeptidoglycan, also known as pseudomurein, and the S-layer is associated with this pseudomurein layer. While crenarchaeal SLPs, such as those found in the extremophile S. islandicus, are generally anchored to the membrane via a C-terminal transmembrane segment [23], most euryarchaeal SLPs appear to be anchored through the covalent attachment of a lipid moiety to their C-terminal end [39][40][41][42]. Euryarchaeal SLPs typically contain a C-terminally located tripartite segment that comprises a highly conserved PGF (proline-glycine-phenylalanine) motif, a transmembrane helix, and a cluster of basic residues.…”

“…Protein lipidation is a further type of post-translational modification in which lipid moieties are covalently linked to proteins. It has been shown to be essential for the attachment of the H. volcanii SLP to the cytoplasmic membrane [39,40,42]. Finally, probably the rarest class of posttranslational modification observed in SLPs is tyrosine phosphorylation.…”

“…Despite the variation in SLP sequences and structures discussed in this article, and despite the vastly different biochemical properties of envelopes in archaea, Gram-positive, and Gramnegative bacteria, some surprising common themes in S-layer biogenesis have been emerging in recent years [39,70,71]. Using real-time optical microscopy and a variety of strategies to fluorescently tag SLPs, the biogenesis of S-layers could be tracked, for the first time, at highresolution in some archaea and bacteria, including H. volcanii, C. crescentus, and C. difficile (Fig.…”