Lipid Anchoring of Archaeosortase Substrates and Midcell 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 morphogenesis 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. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell 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 midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination. IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

The Ribbon-Helix-Helix Domain Protein CdrS Regulates the Tubulin Homolog ftsZ2 To Control Cell Division in Archaea

“…In most bacteria, FtsZ monomers assemble into short filaments that form the cytokinetic ring at midcell, which constricts to divide the mother cell into two daughters of equal size ( 8 – 12 ). FtsZ in archaea appears to function similarly, as previous fluorescence imaging experiments in fixed ( 13 – 16 ) and live ( 17 – 19 ) hypersaline adapted archaeal cells (species Haloferax volcanii [ Hfx. volcanii ]) demonstrated Z-like rings forming at midcell.…”

“…Given the independence of cell elongation from regulation by CdrS and FtsZ2 (Fig. 3 and 5) and that haloarchaeal cells grow by inserting new surface layer (S-layer) material at midcell (17), cell elongation and cytokinesis may occur at the same cell region (Z ring) but may be temporally sequential and separately regulated. CdrS appears to play a key role in coordinating these events.…”

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division

“…In addition, this system can now also be applied to the study of other thermophilic microbes, from eukaryotes to bacteria [ 34 , 35 ]. Although we hope that our work can open new avenues of research and lead further developments in the field of live imaging of thermophiles [ 36 , 37 ], the further development of thermophile cell biology will also depend on the establishment of a thermostable fluorescent proteins [ 38 , 39 ] and the use of microfluidics, to allow for the tracking of cells over long periods across multiple generations, as currently performed in Haloarchaea [ 40 ].…”

The Archaeal Proteome Project advances knowledge about archaeal cell biology through comprehensive proteomics

“…On the other hand, ArtA-dependent processing was compared for H53, ΔartA, and ΔpssA. The ΔpssA mutant FH54 was generated by transforming H53 cells with pFH38 as previously described 95 . In this case, the supernatant and/or membrane fraction of exponentially grown cells have been isolated and used for protease digestion without further fractionation.…”